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.

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.

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.

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.

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.

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.

Genomic characterization and evolution of Tacaiuma orthobunyavirus (Peribunyaviridae family) isolated in Brazil.

Tacaiuma virus (TCMV) is antigenically characterized as a member of the Anopheles A complex in the Orthobunyavirus genus, Peribunyaviridae family (Bunyavirales order). Clinically, the TCMV infection is characterized by acute febrile illness with myalgia and arthralgia lasting three to five days. However, the genomic and evolutionary aspect of this virus has not been elucidated. In this study, we described the complete coding sequences of three segments of two TCMV strains isolated in Brazil and three complete coding sequences of the small segment of three TCMV strains. All the strains sequenced in this study showed the typical genomic organization of orthobunyaviruses that infect vertebrates, except for the absence of the open reading frame that encodes the well-described non-structural small protein. This study presents the genomic and evolutionary characterization of TCMV strains and would be helpful for diagnostic purposes and epidemiology.

A Luminex-based single DNA fragment amplification assay as a practical tool for detecting and serotyping dengue virus.

Dengue is a mosquito-borne viral infection that can evolve from subclinical to severe forms of disease. Early recognition during initial primary and secondary infections correlates with a reduced case-fatality rate in susceptible groups. The aim of this study was to standardize a DNA hybridization assay based on the Luminex technology for detecting and serotyping dengue virus (DENV). Reference DENVs representing the four different serotypes were used as controls to standardize the test. For validation, 16 DENV isolates obtained from a reference laboratory were analyzed in a double-blind manner to validate the test. Sixty blood samples from patients suspected of having dengue fever were used to evaluate the methodology after the validation step, and the results were compared with the reference semi-nested RT-PCR. Additionally, five human samples of each Zika and Chikungunya confirmed patients were used for specificity analysis. The Luminex-based assay correctly identified all 16 DENV isolates. In the evaluation step, the results of the RT-PCR/Luminex assay showed a concordance of 86.7% with those of the semi-nested RT-PCR. None of other virus infection samples was amplified. This is the first description of a hybridization assay that can discriminate the four DENV serotypes using probes against a single DENV sequence. The results indicated that the RT-PCR/Luminex DENV assay designed and evaluated in this study is a valuable additional tool for the early and rapid detection and serotyping of DENV, which could, in the future, be applied to new targets such as the Zika and Chikungunya viruses.

Antibody-enhanced dengue disease generates a marked CNS inflammatory response in the black-tufted marmoset Callithrix penicillata.

Severe dengue disease is often associated with long-term neurological impairments, but it is unclear what mechanisms are associated with neurological sequelae. Previously, we demonstrated antibody-enhanced dengue disease (ADE) dengue in an immunocompetent mouse model with a dengue virus 2 (DENV2) antibody injection followed by DENV3 virus infection. Here we migrated this ADE model to Callithrix penicillata. To mimic human multiple infections of endemic zones where abundant vectors and multiple serotypes co-exist, three animals received weekly subcutaneous injections of DENV3 (genotype III)-infected supernatant of C6/36 cell cultures, followed 24 h later by anti-DENV2 antibody for 12 weeks. There were six control animals, two of which received weekly anti-DENV2 antibodies, and four further animals received no injections. After multiple infections, brain, liver, and spleen samples were collected and tissue was immunolabeled for DENV3 antigens, ionized calcium binding adapter molecule 1, Ki-67, TNFα. There were marked morphological changes in the microglial population of ADE monkeys characterized by more highly ramified microglial processes, higher numbers of trees and larger surface areas. These changes were associated with intense TNFα-positive immunolabeling. It is unclear why ADE should generate such microglial activation given that IgG does not cross the blood-brain barrier, but this study reveals that in ADE dengue therapy targeting the CNS host response is likely to be important.

Emergence and potential for spread of Chikungunya virus in Brazil.

BACKGROUND: In December 2013, an outbreak of Chikungunya virus (CHIKV) caused by the Asian genotype was notified in the Caribbean. The outbreak has since spread to 38 regions in the Americas. By September 2014, the first autochthonous CHIKV infections were confirmed in Oiapoque, North Brazil, and in Feira de Santana, Northeast Brazil. METHODS: We compiled epidemiological and clinical data on suspected CHIKV cases in Brazil and polymerase-chain-reaction-based diagnostic was conducted on 68 serum samples from patients with symptom onset between April and September 2014. Two imported and four autochthonous cases were selected for virus propagation, RNA isolation, full-length genome sequencing, and phylogenetic analysis. We then followed CDC/PAHO guidelines to estimate the risk of establishment of CHIKV in Brazilian municipalities. RESULTS: We detected 41 CHIKV importations and 27 autochthonous cases in Brazil. Epidemiological and phylogenetic analyses indicated local transmission of the Asian CHIKV genotype in Oiapoque. Unexpectedly, we also discovered that the ECSA genotype is circulating in Feira de Santana. The presumed index case of the ECSA genotype was an individual who had recently returned from Angola and developed symptoms in Feira de Santana. We estimate that, if CHIKV becomes established in Brazil, transmission could occur in 94% of municipalities in the country and provide maps of the risk of importation of each strain of CHIKV in Brazil. CONCLUSIONS: The etiological strains associated with the early-phase CHIKV outbreaks in Brazil belong to the Asian and ECSA genotypes. Continued surveillance and vector mitigation strategies are needed to reduce the future public health impact of CHIKV in the Americas.

Polymorphism of DC-SIGN (CD209) promoter in association with clinical symptoms of dengue fever.

C-type lectin DC-SIGN receptor, encoded by CD209, plays a key role in the infection of dendritic cells by dengue virus (DENV). Because the -336A/G SNP (rs4804803) polymorphism in the promoter of CD209 modulates DC-SIGN expression, we investigated the putative association of this polymorphism with DENV infection and its pathogenesis. A control sample of 72 individuals, rigorously selected through a clinical investigation for absence of past dengue fever (DF) was compared to a sample of 168 patients (156 classical DF; 12 dengue hemorrhagic fever), all residents from Pará, Brazil. However, the prevalence of symptoms showed a trend higher in the AA genotype (Wilcoxon test; Z=2.02; p=0.04). Hence, our findings indicate that the G allele downregulates the spectrum of symptoms during the early acute phase of DENV infection, putatively decreasing the viremia, as suggested in the literature.

Phylogeography of dengue virus serotype 4, Brazil, 2010-2011.

Dengue virus serotype 4 (DENV-4) reemerged in Roraima State, Brazil, 28 years after it was last detected in the country in 1982. To study the origin and evolution of this reemergence, full-length sequences were obtained for 16 DENV-4 isolates from northern (Roraima, Amazonas, Pará States) and northeastern (Bahia State) Brazil during the 2010 and 2011 dengue virus seasons and for an isolate from the 1982 epidemic in Roraima. Spatiotemporal dynamics of DENV-4 introductions in Brazil were applied to envelope genes and full genomes by using Bayesian phylogeographic analyses. An introduction of genotype I into Brazil from Southeast Asia was confirmed, and full genome phylogeographic analyses revealed multiple introductions of DENV-4 genotype II in Brazil, providing evidence for >3 introductions of this genotype within the last decade: 2 from Venezuela to Roraima and 1 from Colombia to Amazonas. The phylogeographic analysis of full genome data has demonstrated the origins of DENV-4 throughout Brazil.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus).

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Full-length sequencing and genetic characterization of Breu Branco virus (Reoviridae, Orbivirus) and two related strains isolated from Anopheles mosquitoes.

Breu Branco virus (BE AR 492347) was isolated from Anopheles (Nyssorhynchus) triannulatus mosquitoes captured in Tucuruí, Pará State, northern Brazil, in 1988. No cross-reactivity by complement-fixation tests was observed between Breu Branco virus and other known arboviruses. Results of electron microscopy and physicochemical tests suggested that Breu Branco virus may be a member of the family Reoviridae. In order to elucidate its taxonomic status, a comprehensive genetic characterization was conducted for Breu Branco virus and related strains (BE AR 494475 and BE AR 486204) that were also isolated from Anopheles mosquitoes in the same area. This included full-length genome sequencing, determination of genetic traits and phylogenetic analysis. Breu Branco virus showed a similar genome organization to members of the genus Orbivirus, family Reoviridae. Genetically, Breu Branco virus was indistinguishable from strains BE AR 494475 and BE AR 486204. Phylogenetic analysis suggested that Breu Branco virus BE AR 492347 and its related strains constitute a novel species of the genus Orbivirus. Breu Branco virus is the first Brazilian orbivirus and the fifth orbivirus in the world to be sequenced fully.

Dengue virus type 3 in Boa Vista, Northern Brazil

Casos de doença febril aguda ocorridos em Boa Vista, estado de Roraima, Brasil foram investigados em julho de 2005. Amostras de sangue (n igual a 142) foram obtidas de pacientes clinicamente suspeitos de febre do dengue (FD). A tentativa de isolamento viral foi realizada em células C6/36 para pacientes com menos de cinco dias de doença (n igual a 96). As cepas isoladas foram identificadas pelo teste de imunofluorescência indireta (IFI) utilizando anticorpos monoclonais, bem como pela técnica de RT-PCR. Amostras de soro foram testadas pelo método de inibição da hemaglutinação (IH) e confirmadas pelo IgM-ELISA. O vírus dengue 3 (VDEN-3) foi isolado e detectado po RT-PCR em 41 pacientes. A região E de sete cepas foi sequenciada, sendo as mesmas identificadas como pertencentes ao genótipo III. Pelo teste de IH, 98 amostras de soros foram positivas para anticorpos IH para o vírus da Dengue, sendo os resultados confirmados pela detecção de anticorpos IgM para dengue. Clinicamente, todos os pacientes mostraram quadro de FD, sendo todas as faixas etárias afetadas independentes do sexo. A epidemia de dengue ocorrida em Roraima foi causada pelo VDEN-3, genótipo III, genótipo este que circula nas Américas desde 1994.