The NLRP3 inflammasome is involved with the pathogenesis of Mayaro virus.

Mayaro virus (MAYV) is an arbovirus that circulates in Latin America and is emerging as a potential threat to public health. Infected individuals develop Mayaro fever, a severe inflammatory disease characterized by high fever, rash, arthralgia, myalgia and headache. The disease is often associated with a prolonged arthralgia mediated by a chronic inflammation that can last months. Although the immune response against other arboviruses, such as chikungunya virus (CHIKV), dengue virus (DENV) and Zika virus (ZIKV), has been extensively studied, little is known about the pathogenesis of MAYV infection. In this study, we established models of MAYV infection in macrophages and in mice and found that MAYV can replicate in bone marrow-derived macrophages and robustly induce expression of inflammasome proteins, such as NLRP3, ASC, AIM2, and Caspase-1 (CASP1). Infection performed in macrophages derived from Nlrp3-/-, Aim2-/-, Asc-/-and Casp1/11-/-mice indicate that the NLRP3, but not AIM2 inflammasome is essential for production of inflammatory cytokines, such as IL-1ß. We also determined that MAYV triggers NLRP3 inflammasome activation by inducing reactive oxygen species (ROS) and potassium efflux. In vivo infections performed in inflammasome-deficient mice indicate that NLRP3 is involved with footpad swelling, inflammation and pain, establishing a role of the NLRP3 inflammasome in the MAYV pathogenesis. Accordingly, we detected higher levels of caspase1-p20, IL-1ß and IL-18 in the serum of MAYV-infected patients as compared to healthy individuals, supporting the participation of the NLRP3-inflammasome during MAYV infection in humans.

Development of an Enzyme-Linked Immunosorbent Assay To Detect Antibodies Targeting Recombinant Envelope Protein 2 of Mayaro Virus.

Mayaro virus (MAYV) is a neglected arthropod-borne virus (arbovirus) antigenically clustered into the Semliki Forest complex group of Alphavirus genus (Togaviridae family), maintained in an unclear zoonotic cycle involving mosquitoes from Haemagogus genus as the main vector. The genome is composed of a positive single-stranded RNA of 11.5 kb in length, which contains two genes that encode four nonstructural (nsP1 to nsP4) and five structural (C, E3, E2, 6K, and E1) proteins. In the present study, we have developed an enzyme-linked immunosorbent assay (ELISA) using as antigen the recombinant envelope protein 2 of MAYV produced in an Escherichia coli system (rE2-MAYV ELISAs). A panel of 68 human serum samples from suspected arboviral cases was analyzed and titrated for anti-MAYV IgM and IgG antibody detection. The rE2-MAYV ELISA detected 33.8% (23/68) IgG-positive samples, demonstrating 100% sensitivity and 78.95% specificity compared to the MAYV-specific 50% plaque reduction neutralization assay. In addition, the positive MAYV-neutralizing samples showed high titers of detection by rE2-MAYV ELISA, suggesting a highly sensitive test. The rE2-MAYV ELISA also detected 42.5% (29/68) IgM-positive samples, of which 13.8% (4/29) presented high-avidity interactions with rE2-MAYV. Cross-reactivity was observed with Chikungunya virus (CHIKV)-specific murine antibody sample but not with CHIKV-specific human and other Alphavirus murine antibodies. In short, we have developed a rapid, simple, specific, and sensitive MAYV rE2-ELISA, and our preliminary results show its potential applicability to diagnosis of MAYV infections.

Mayaro virus and dengue virus 1 and 4 natural infection in culicids from Cuiabá, state of Mato Grosso, Brazil.

This study aimed to verify the diversity of Culicidae species and their frequency of infection with flaviviruses and alphaviruses in Cuiabá, state of Mato Grosso, Brazil. Mosquitoes were captured with Nasci aspirators and hand net in 200 census tracts, identified alive at species level and pooled in one-20 (11,090 mosquitoes, 14 species). Female pools (n = 610) were subjected to multiplex seminested-reverse transcription-polymerase chain reaction (RT-PCR) for 11 flavivirus and five alphavirus. Positive pools were tested by single RT-PCR followed by nucleotide sequencing, by RT-PCR for E1 gene [Mayaro virus (MAYV)] and by inoculation in Vero cells (MAYV) or C6/36 cells (flaviviruses). One/171 Aedes aegypti was positive for dengue virus (DENV)-1, 12/403 Culex quinquefasciatus, and four/171Ae. aegypti for MAYV, which was isolated from two pools containing two nonengorged females of Ae. aegypti and two of Cx. quinquefasciatus. DENV-4 was detected in 58/171 pools of Ae. aegytpi, 105/403 Cx. quinquefasciatus, two/five Psorophora sp., two/11 Psorophora varipes/Psorophora albigenu, one/one Sabethes chloropterus, two/five Culex bidens/Culex interfor, and one/one Aedes sp. DENV-4 was isolated from two pools containing three and 16 nonengorged Cx. quinquefasciatus females. Phylogenetic analysis revealed MAYV belongs to genotype L, clustering with human samples of the virus previously identified in the city. Cuiabá has biodiversity and ecosystem favourable for vector proliferation, representing a risk for arbovirus outbreaks.

Detection of Oropouche virus segment S in patients and inCulex quinquefasciatus in the state of Mato Grosso, Brazil.

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatus captured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatus pools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

Molecular detection of Mayaro virus during a dengue outbreak in the state of Mato Grosso, Central-West Brazil.

Mayaro virus (MAYV) is frequently reported in Pan-Amazonia. The aim of this study was to investigate the circulation of alphaviruses during a dengue outbreak in the state of Mato Grosso, Brazil. Serum samples from dengue-suspected patients were subjected to multiplex semi-nested reverse transcriptase polymerase chain reaction for 11 flaviviruses and five alphaviruses, to nucleotide sequencing and to viral isolation. MAYV was detected in 15 (2.5%) of 604 patients. Twelve were co-infected with dengue virus 4, which was isolated from 10 patients. The molecular detection of MAYV in dengue-suspected patients suggests that other arboviruses may be silently circulating during dengue outbreaks in Brazil.

Associations between epidemiological and laboratory parameters and disease severity in hospitalized patients with COVID-19 during first and second epidemic waves in middle south Mato Grosso.

BACKGROUND: COVID-19 is a multisystemic disease characterized by respiratory distress. Disease severity is associated with several factors. Here we characterize virological findings and evaluate the association of laboratorial, epidemiological, virological findings and clinical outcomes of 251 patients during the first and second epidemic waves of COVID-19. METHODS: This transversal study used biological samples and data from patients hospitalized with COVID-19 between May 2020 and August 2021 in the metropolitan region of Cuiabá, Mato Grosso Brazil. Biological samples were subjected to RT-qPCR and MinION sequencing. Univariate and multivariate logistic regression and Odds ratio were used to correlate clinical, laboratorial, epidemiological data. FINDINGS: Patients were represented by males (61.7%) with mean age of 52.4 years, mild to moderate disease (49,0%), overweight/obese (69.3%), with comorbidities (66.1%) and evolving to death (55.38%). Severe cases showing symptoms for prolonged time, ≥ 25% of ground-glass opacities in the lungs and fatality rate increased significantly in second wave. Fatality was statistically associated to > 61 years of age,>25% ground-glass opacities in the lungs, immune, cardiac, or metabolic comorbidities. Higher viral load (p < 0.01/p = 0.02 in each wave), decreased erythrocyte (p < 0.01), hemoglobin (p < 0.05/p < 0.01), hematocrit (p < 0.01), RDW (p < 0.01), lymphocyte (p < 0.01), increased leucocyte (p < 0.01), neutrophil (p < 0.01) and CRP levels (p < 0.01) showed significant association with fatality in both waves, as did Neutrophil/Platelet (NPR; p < 0.01), Neutrophil/Lymphocyte (NLR; p < 0.01) and Monocyte/Lymphocyte ratio (MLR; p < 0.01). SARS-CoV-2 genomes from lineage B.1.1.33(n = 8) and Gamma/P.1(n = 15) shared 6/7 and 20/23 lineage-defining mutations, respectively. MAIN CONCLUSIONS: Severity and mortality of COVID-19 associated with a panel of epidemiological and laboratorial findings, being second wave, caused by Gamma variant, more severe in this in-hospital population.

Investigation of Yellow Fever Virus at the Human-Animal Interface after a Zika Virus Outbreak in Midwest Brazil.

Following the first report of zika virus in March 2015, Brazil experienced its largest sylvatic yellow fever outbreak between 2016 and 2019. This study aimed to investigate the circulation of yellow fever virus (YFV) in non-human primates (NHPs) and mosquitoes collected in urban parks and other metropolitan areas of midwest Brazil between 2017 and 2018. Whole blood samples from 80 NHPs, including 48 black-tailed marmosets (Mico melanurus) and 2332 mosquitoes from six different genera, were collected in the states of Mato Grosso (MT) and Mato Grosso do Sul (MS) and then tested for YFV by RT-qPCR. Additionally, 23 plasma samples of NHPs were tested for neutralizing antibodies for YFV by a plaque reduction neutralization test (PRNT). No YFV RNA or neutralizing antibodies for YFV were detected in NHPs and mosquitoes from MT and MS. The continuous monitoring of YFV circulation in different species of NHPs and vectors in urban areas is instrumental to quickly assess potentially unknown maintenance cycles of yellow fever at the human-animal interface in Brazil.

Disclosing the virome of Aedes, Anopheles and Culex female mosquitoes, Alto Pantanal of Mato Grosso, Brazil, 2019.

Using Illumina NextSeq sequencing and bioinformatics, we identified and characterized thirty-three viral sequences of unsegmented and multipartite viral families in Aedes spp., Culex sp. and Anopheles darlingi female mosquito pools from Porto São Luiz and Pirizal, Alto Pantanal. Seventeen sequences belong to unsegmented viral families, twelve represent putative novel insect-specific viruses (ISVs) within families Chuviridae (3/33; partial genomes) and coding-complete sequences of Xinmoviridae (1/33), Rhabdoviridae (2/33) and Metaviridae (6/33); and five coding-complete sequences of already-known ISVs. Notably, two putative novel rhabdoviruses, Corixo rhabdovirus 1 and 2, were phylogenetically related to Coxipo dielmovirus, but separated from other Alpharhabdovirinae genera, sharing Anopheles spp. as host. Regarding multipartite families, sixteen segments of different putative novel viruses were identified (13 coding-complete segments) within Durnavirales (4/33), Elliovirales (1/33), Hareavirales (3/33) and Reovirales (8/33) orders. Overall, this study describes twenty-eight (28/33) putative novel ISVs and five (5/33) already described viruses using metagenomics approach.

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.

Inflammation, fibrosis and E1 glycoprotein persistence in joint tissue of patients with post-Chikungunya chronic articular disease.

INTRODUCTION: Chikungunya chronic joint disease causes debilitating arthralgia, significantly impacting the quality of life of affected individuals. METHODS: In this study, patients underwent clinical follow-ups, joint biopsies, and pre-biopsy and 24 months post-biopsy serum dosage of cytokines. RESULTS: All participants were female and had pain in 12 joints on average, with 41.17% exhibiting moderate disease activity. Histopathological analysis revealed collagen deposition. Indirect immunofluorescence detected the CHIKV glycoprotein E1 antigen, and an increase in cytokines. CONCLUSIONS: Persistent inflammation and ineffective antiviral immune responses leading to antigen persistence may contribute to chronic CHIKV arthritis.

Diagnostic and vaccine potential of Zika virus envelope protein (E) derivates produced in bacterial and insect cells.

Introduction: In the present study we evaluated the features of different recombinant forms of Zika virus (ZIKV) proteins produced in either bacterial (Eschericha coli) or insect cells (Drosophila melanogaster). The ZIKV-envelope glycoprotein (EZIKV) is responsible for virus entry into host cells, is the main target of neutralizing antibodies and has been used as a target antigen either for serological tests or for the development of subunit vaccines. The EZIKV is composed of three structural and functional domains (EDI, EDII, and EDIII), which share extensive sequence conservation with the corresponding counterparts expressed by other flaviviruses, particularly the different dengue virus (DENV) subtypes. Methods: In this study, we carried out a systematic comparison of the antigenicity and immunogenicity of recombinant EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells. For the antigenicity analysis we collected 88 serum samples from ZIKV-infected participants and 57 serum samples from DENV-infected. For immunogenicity, C57BL/6 mice were immunized with two doses of EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells to evaluate humoral and cellular immune response. In addition, AG129 mice were immunized with EZIKV and then challenge with ZIKV. Results: Testing of samples collected from ZIKV-infected and DENV-infected participants demonstrated that the EZIKV and EDIIIZIKV produced in BL21 cells presented better sensitivity and specificity compared to proteins produced in S2 cells. In vivo analyses were carried out with C57BL/6 mice and the results indicated that, despite similar immunogenicity, antigens produced in S2 cells, particularly EZIKV and EDIIIZIKV, induced higher ZIKV-neutralizing antibody levels in vaccinated mice. In addition, immunization with EZIKV expressed in S2 cells delayed the onset of symptoms and increased survival rates in immunocompromised mice. All recombinant antigens, either produced in bacteria or insect cells, induced antigen-specific CD4+ and CD8+ T cell responses. Conclusion: In conclusion, the present study highlights the differences in antigenicity and immunogenicity of recombinant ZIKV antigens produced in two heterologous protein expression systems.

Chikungunya, Zika, Mayaro, and Equine Encephalitis virus detection in adult Culicinae from South Central Mato Grosso, Brazil, during the rainy season of 2018.

INTRODUCTION: Several arboviruses causing human disease have been reported in Brazil. In nature, arboviruses maintain a lifecycle involving vertebrates and vectors, which may contribute for periodical reemergence of those of public health concern in tropical regions, as Mato Grosso State (MT). In this study, we searched for arboviruses in mosquito body pools sampled during the rainy season of 2018 in 21 bird watching points of Cuiabá and Varzea Grande, South Central MT. METHODS: In total, 2873 (57%) males and 2167 (43%) females belonging to six urban and sylvatic mosquito genera allocated to 398 pools were subjected to RNA extraction and RT-PCR for arboviruses. Positive pools were subjected to virus isolation in C6/36 cells. RESULTS: A total of 102/398 pools, 66/233 (29.6%) of females, and 36/165 (21.8%) of males, mostly sampled in May (31/102), were positive for arboviruses. Chikungunya virus (CHIKV) was distributed in 19 points, Zika virus (ZIKV) was found in 14 points, Mayaro virus (MAYV) in 10 points, and East Equine encephalitis virus (EEEV) in three points. Culex quinquefasciatus pools (39/89 of females and 24/99 of males) were positive for CHIKV, ZIKV, and MAYV; Aedes (Stg) aegypti pools (11/46 of females and 12/33 of males) for CHIKV, ZIKV, MAYV, and EEEV; Aedes albopictus female pools (8/29) for CHIKV, ZIKV, and EEEV; and Psorophora albigenu (2/12) and Psorophora ferox female pools (4/16) for CHIKV. CONCLUSIONS: Arbovirus molecular detection in mosquito populations varies considerable between geographical regions and epidemics, influenced by genetic characteristics and microbiome interference on virus replication. Although infected females are responsible for the transmission to vertebrates during bloodfeeding, male infection by CHIKV, ZIKV, and MAYV resultant from vertical route could lead to interepidemic maintenance of these arboviruses in their natural reservoirs.

Regional mutations in CHIKV-ECSA genomes and detection of other viruses in the serum of acute febrile patients by a metagenomic approach in Mato Grosso, Central-Western Brazil, 2018.

Mato Grosso (MT) State is part of central western Brazil and has a tropical permissive environment that favors arbovirus outbreaks. A metagenomic approach was used to identify viral genomes in seven pools of serum from patients (n=65) with acute febrile disease. Seven chikungunya virus (CHIKV) genomes were determined, showing four amino acid changes found only in CHIKV genomes obtained in MT since 2018: nsP2:T31I, nsP3: A388V, E3:T201I and E3:H57R, in addition to other mutations in E1, nsP2 and nsP4. Six parvovirus B19 (B19V) genotype I genomes (4771-5131 nt) showed four aa alterations (NS1:N473D, R579Q; VP1:I716T; and 11 kDa:V44A) compared to most similar B19V from the USA. Coinfection between CHIKV and B19V was evidenced in 22/65 (33.8%) patients by RT‒PCR and PCR, respectively. Other viruses found in these pools include human pegivirus C, torque teno virus 3, an unclassified TTV and torque teno mini virus. Metagenomics represents a useful approach to detect viruses in the serum of acute febrile patients suspected of arbovirus disease.

Neurological infection by chikungunya and a triple Arbovirus co-infection in Mato Grosso, Central Western Brazil during 2019.

BACKGROUND: Neurological viral infection is frequently associated to enterovirus, herpesvirus and arboviruses. These infections may cause severe clinical outcomes, long lasting sequelae or death. Few studies have addressed viral neurological infections etiology in Brazil. OBJECTIVES: Identification of viruses in the cerebral spinal fluid (CSF) of human neurological infections suspected of viral etiology during January and May 2019 in Midwestern Brazil. MATERIALS AND METHODS: Clinical, laboratory and epidemiological information was gathered from medical records. In addition, an aliquot of the sampled CSF was subjected to viral RNA/DNA extraction, randomic dscDNA amplification by PCR, DNA purification and Ilumina HiSeq 2500 sequencing. RESULTS: Six viral genomes belonging to Chikungunya virus (CHIKV) East-Central-South African (ECSA) genotype (10.834-11.804 nt in length) confirmed lately by RT-PCR for CHIKV envelope were present in all six liquor samples. These genomes present two mutations, nsP2:T31I and nsP3:A388V, shared with other Mato Grosso State strains from 2019, not present in sequences of the virus from previous years obtained in the State. One case was a triple co-infection also confirmed through RT-PCR, with Dengue virus serotype 4 genotype II (NS5; 874 nt) and Oropouche virus genotype IA (segment S; 302 nt). CSF was clear and colorless (5/6 patients), with >10% of lymphomononuclear cells (6/6), 1-99 erythrocytes/mm3 (5/6), glucose levels >50 mg/dl (4/5) e > 10 mg/dl of proteins (4/4). One patient evolved to death, and another, a newborn, presented sequelae after recovery. CONCLUSIONS: Despite herpesviruses and enteroviruses are frequent etiologies of neurological infections, the casuistic here reported was associated to arboviruses already known to be responsible for acute febrile illness outbreaks in the state of Mato Grosso, Midwestern Brazil.

Clinical and genomic data of sars-cov-2 detected in maternal-fetal interface during the first wave of infection in Brazil.

Brazil has the highest SARS-CoV-2 case-fatality rate in pregnant women in the Americas. In this study, clinical and virological findings of five mildly symptomatic pregnant women and their infected fetuses/newborns treated at a referral hospital for COVID19-pregnant women in Midwestern Brazil are reported. Mother and fetal samples were tested by RT-qPCR, ECLIA and Illumina MiSeq sequencing. From the five cases, one resulted in spontaneous abortion, one was stillborn, two were preterm births and one full-term birth. Maternal and fetal placenta, newborn and stillborn secretions were SARS-CoV-2+; one neonate developed ground-glass opacities in his lungs. One neonate's umbilical cord was IgG+ and all were IgM negative upon hospital discharge. Genomes recovered from two placentas belong to the B.1.1.28 and B.1.1.33 lineages and present nonsynonymous mutations associated with virus fitness and infectivity; other not frequently reported mutations (B.1.1.33: NSP3 V2090G, M A2S and ORF3ab S253P and Y264N; B.1.1.28: NSP3 E995D, NSP12 R240K, NSP14H1897Y and in ORF7b V21F) were found in proteins involved in viral replication, viral induction of apoptosis, viral interference on interferon and on NF-Κß pathways. Phylogeny indicates the south of Brazil as the possible origin of these lineages circulating in Mato Grosso State. These findings contribute to describe SARS-CoV-2 infection and outcomes in pregnant women and their fetuses, at any stage of gestation and even in mild symptomatic cases.

A Novel Host of an Emerging Disease: SARS-CoV-2 Infection in a Giant Anteater (Myrmecophaga tridactyla) Kept Under Clinical Care in Brazil.

A young male free-ranging giant anteater (Myrmecophaga tridactyla) was found with paralysis of pelvic limbs on a highway and kept under human care. Radiographs confirmed multiple incomplete fractures in the thoracolumbar vertebrae. Due to the poor prognosis, euthanasia was chosen. The infection was established by viral SARS-CoV-2 RNA detection in the rectal swab, spleen and kidney samples. Immunohistochemistry detected the viral nucleocapsid protein in sections of the lungs, liver, spleen, lymph nodes, and large intestine sections, and spike protein antigen in the lung tissue. Pilosa order species should be included as potential hosts of natural infection of SARS-CoV-2.

Persistent SARS-CoV-2 antigen presence in multiple organs of a naturally infected cat from Brazil.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the disease coronavirus 2019 (COVID-19) in humans. SARS-CoV-2 has been identified in cats with or without clinical signs. Case presentation: We describe the pathological and molecular findings in a six-month-old asymptomatic cat with SARS-CoV-2 infection from Brazil, belonging to a human family with COVID-19 cases. The pool of nasopharynx and oropharynx swabs at day zero tested positive by RT-qPCR for SARS-CoV-2. No amplification resulted from molecular testing performed on days 7 and 14. The cat was hit by a car and died 43 days after the molecular diagnosis. Immunohistochemistry at post-mortem examination demonstrated nucleocapsid protein in samples from the lungs, kidneys, nasal conchae, trachea, intestine, brain and spleen. Conclusion: The present study has highlighted the possibility that viral antigens can be detected by immunohistochemistry in multiple organs six weeks after infection, although the same tissues tested negative by RT-PCR.

Sialovirome of Brazilian tropical anophelines.

Anophelinae is a widely dispersed Culicidae subfamily that may carry a unique virome. Here we herein report the set of viruses found in 323 salivary glands of 16 anopheline species sampled at Upper Pantanal, Chapada dos Guimarães National Park and Coxipó river basin, South Central Mato Grosso, Brazil, pooled (n = 11) and subjected to high throughput sequencing. Metagenomics revealed the presence of nine viral sequences belonging to novel viruses from seven viral families: Purunga is a putative novel orbivirus sharing 74% and 65% aa identity, respectively, with the VP1 and VP3 segments of Changuinola serogroup, Jaracatiá flavivirus shares 60% amino-acid (aa) identity with Aedes flavivirus. Coxipó dielmovirus and Chapada dielmovirus shared 51% and 39% aa identity with Merida virus. Coloiado-orthomyxo like virus is 57.1-64.8% identical at aa level to Aedes albonnulatus orthomyxo-like virus. Mujica picorna-like virus shares 49% aa identity with Flen picorna-like virus and Chiquitos virus is 50% similar to Ista virus, both from Picornavirales order. Cerrado partiti-like-virus shares 75-86% aa identity with Atrato partiti-like virus 2. We also found the S and L segments of Anopheles triannulatus orthophasmavirus (92% identity) in Anopheles lutzi from Chapada dos Guimarães. The identification of these putative novel viruses underscore the wide dispersion of viruses in culicid hosts contributing to extensions on mosquito virome descriptions.

Influence of climatic variables on the Aedes aegypti and Culex quinquefasciatus populations in Mato Grosso, Brazil.

INTRODUCTION: Aedes aegypti and Culex quinquefasciatus are vector species responsible for the transmission of important arboviruses. METHODS: Adult mosquitoes were collected in the urban areas of four municipalities in Mato Grosso within 1 year. RESULTS: A total of 19,110 mosquitoes were collected. Among them, 16,578 (86,8%) were C. quinquefasciatus (44% female and 56% male); 2,483 (13%), A. (Stegomyia) aegypti (54% female and 46% male); and 49 (0,30%), from the genus Psorophora, Anopheles, Coquilettidia, and Sabethes. A significant correlation was observed between the number of mosquitoes from all species and dew point (female mosquitoes, p = 0.001; male mosquitoes, p = 0.001). CONCLUSIONS: The results of this study may be used as environmental indicators of mosquito populations.

Insect-specific viruses and arboviruses in adult male culicids from Midwestern Brazil.

Viruses were identified from male anthropophilic mosquitoes from Mato Grosso (MT) State, Midwest Brazil from February 2017 to January 2018. Mosquitoes tested included Aedes (Stegomyia) aegypti (1139 males; 84 pools), Culex quinquefasciatus (9426 males; 179 pools), Culex sp. (3 males; 3 pools) and Psorophora albigenu (1 male; 1 pool) collected from four cities of MT. Pools were subjected to viral RNA extraction followed by RT-PCRs specific for ten flaviviruses, five alphaviruses and Simbu serogroup of orthobunyaviruses. Positive pools were passaged three times in VERO cells (alphavirus and orthobunyavirus) or C6/36 cells (flavivirus), with isolates confirmed through RT-PCR and nucleotide sequencing. We detected pools positive for Ilhéus (1 pool), dengue serotype 4 (1), Mayaro (12), equine encephalitis virus (1) yellow fever (1), Oropouche (2), Zika (4) and chikungunya (12) viruses. High throughput sequencing of arbovirus positive pools identified 35 insect-specific viruses (ISVs) from the families Circoviridae (2), Parvoviridae (2), Totiviridae (1), Flaviviridae (1), Iflaviridae (2), Mesoniviridae (4), Nodaviridae (2), Luteoviridae (1), Phasmaviridae (1) Phenuiviridae (2), Rhabdoviridae (2), Orthomyxoviridae (1), Xinmoviridae (1), and unclassified Bunyavirales (1), unclassified Picornavirales (3), unclassified Riboviria (4) and taxon Negevirus (5). From these, five novel viruses were tentatively named Mojica circovirus, Kuia iflavirus, Muxirum negevirus, Lambada picorna-like virus and Tacuru picorna-like virus. Our findings underscore the diversity and wide geographical distribution of ISVs and arboviruses infecting male culicids.