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State of Sao Paulo Research Foundation (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2013/15313-4]

Background Phylogenetic studies indicate bats as original hosts of SARS-CoV-2. However, it remains unclear whether other animals, including pets, are crucial in the spread and maintenance of COVID-19 worldwide. Methods In this study, we analyzed the first fatal case of a SARS-CoV-2 and FeLV co-infection in an eight-year-old male cat. We carried out a clinical evaluation and several laboratory analyses. Results As main results, we observed an animal presenting severe acute respiratory syndrome and lesions in several organs, which led to the animal’s death. RT-qPCR analysis showed a SARS-CoV-2 as the causative agent. The virus was detected in several organs, indicating a multisystemic infection. The virus was found in a high load in the trachea, suggesting that the animal may have contribute to the transmission of the virus. The whole-genome sequencing revealed an infection by SARS-CoV-2 Gamma VOC (P.1), and any mutations indicating host adaptation were observed. Conclusion Our data show that FeLV-positive cats are susceptible to SARS-CoV-2 infection and raise questions about the potential of immunocompromised FeLV-positive cats to act as a reservoir for SARS-CoV-2 new variants.

Background: Coronaviruses are recognized for their ability to cross the species barrier and infect new hosts. The coronavirus disease 2019 (COVID-19) is caused by the new coronavirus SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). It remains unclear whether other animals, including pets, are crucial in the spread and maintenance of COVID-19 worldwide. Methods: In this study, we analysed the first fatal case of a SARS-CoV-2 and FeLV (Feline leukemia virus) co-infection of an eight-year-old male cat. We carried out a clinical evaluation, pathological analysis, and viral genomic analysis. Results: As main results, we observed an animal presenting severe acute respiratory syndrome and lesions in several organs, which led to animal’s death. The causative agent was confirmed to be SARS-CoV-2, variant of concern P.1 (Gamma). The virus presented a pattern of mutations potentially associated with feline infection. In addition, the virus was detected by RT-qPCR in the spleen, liver, heart, lungs, trachea, intestines and kidneys, indicating a multisystemic viral infection. The virus was found in a high load in the trachea, suggesting a capacity of transmitting the virus. Conclusion: Our data show that felines, such as FeLV-positive cats, are susceptible to SARS-CoV-2 infection, and may be intermediate hosts in this pandemic.

Aedes (Stegomyia) aegypti (A. aegypti) transmits arboviral diseases of high public health importance, including those caused by Zika virus (ZIKV), Dengue virus (DENV), Chikungunya virus (CHIKV) and Yellow fever virus (YFV). Barreiras is a city with 157,638 inhabitants in the West of the State of Bahia, Northeast of Brazil. The climate is dry, with well-determined and concentrated seasons of rains. The city is crossed by a Federal Highway and by the Rio Grande river. In this study, we aimed to understand the dynamics of mosquito vectors and arboviral diseases in Barreiras. We used correlation statistics to investigate a possible relationship among rains, mosquito abundance and transmission of diseases. In addition, as a preliminary population genetics estimate, we used geometric morphometrics to compare mosquitoes from areas limited by a highway and a river. We found that i) infestation occurs in rain-dependent cycles and that ii) both, the river and the highway segregate populations of A. aegypti in different areas of the studied city. Our results indicate that it is necessary to treat anthropic containers with mosquito breading capacity during both, the dry and rain seasons in urban areas similar to Barreiras.

Background The arboviruses Zika virus (ZIKV) and Dengue virus (DENV) have important epidemiological impact in Brazil and other tropical regions of the world. Recently, it was shown that previous humoral immunity to DENV enhances ZIKV replication in vitro, which may lead to more severe forms of the disease. Thus, traditional approaches of vaccine development aiming to control viral infection through neutralizing antibodies may induce cross-reactive enhancing antibodies. In contrast, cellular immune response was shown to be capable of controlling DENV infection independently of antibodies. The aim of the present study was to design a flavivirus NS5 protein capable of inducing a cellular immune response against DENV and ZIKV. Methods A consensus sequence of ZIKV NS5 protein was designed among isolates from various continents. Epitopes were predicted for the most prevalent alleles of class I and II HLA in the Brazilian population. Then, this epitopes were analyzed with regard to their conservation, population coverage and distribution along the whole antigen. Results Nineteen epitopes predicted to be more reactive (percentile rank

The field of morphometrics is developing quickly. Recent advances have enabled geometric techniques to be applied to many zoological problems, particularly those involving epidemiologically-relevant mosquitoes. Herein, we briefly introduce geometric morphometric (GM) techniques and then review selected groups of mosquitoes (Culicidae) to which those techniques have been applied. In most of the reviewed cases, GM was capable of satisfactorily discriminating among the tested groups primarily when the studies considered differences within and among species, sexual dimorphism, treatments and the separation of laboratory strains. Although GM approaches have developed quite rapidly, some caution must be taken during data processing for a reliable biometrical approach, such as allometry and asymmetry analyses, scale removal and wing clarification staining for landmark digitization. We also critically forecast directions in this field and discuss how the creation of image databases should enhance species identification in culicids.

Culicidae mosquitoes are important vectors of pathogens; however, males and females have different responses to selective pressures, leading to distinct evolutionary outcomes. A better understanding of this phenomenon is crucial to establish effective control strategies for this mosquito. Although Aedes fluviatilis is considered a secondary vector for several diseases, there is a dearth of data about its ecology, population structure and sexual dimorphism. The goal of this study was therefore to assess sexual dimorphism patterns in Aedes fluviatilis. Wings from males and females were analyzed both by geometric morphometrics. Pairwise cross-validated reclassification tests indicated significant sexual dimorphism between specimens from each gender. Canonical variation analysis showed significant morphometrical differences between genders, indicating that sexual dimorphism might be a result of evolutionary pressures in this species and may be useful when developing control strategies for this mosquito.

Although native to the tropical and subtropical areas of Southeast Asia, Aedes albopictus is now found on five continents, primarily due to its great capacity to adapt to different environments. This species is considered a secondary vector of dengue virus in several countries. Wing geometric morphometrics is widely used to furnish morphological markers for the characterisation and identification of species of medical importance and for the assessment of population dynamics. In this work, we investigated the metric differentiation of the wings of Ae. albopictus samples collected over a four-year period (2007-2010) in São Paulo, Brazil. Wing size significantly decreased during this period for both sexes and the wing shape also changed over time, with the wing shapes of males showing greater differences after 2008 and those of females differing more after 2009. Given that the wings play sex-specific roles, these findings suggest that the males and females could be affected by differential evolutionary pressures. Consistent with this hypothesis, a sexually dimorphic pattern was detected and quantified: the females were larger than the males (with respect to the mean) and had a distinct wing shape, regardless of allometric effects. In conclusion, wing alterations, particularly those involving shape, are a sensitive indicator of microevolutionary processes in this species.

Mosquitoes are responsible for the transmission of important infectious diseases, causing millions of deaths every year and endangering approximately 3 billion people around the world. As such, precise identification of mosquito species is crucial for an understanding of epidemiological patterns of disease transmission. Currently, the most common method of mosquito identification relies on morphological taxonomic keys, which do not always distinguish cryptic species. However, wing geometric morphometrics is a promising tool for the identification of vector mosquitoes, sibling and cryptic species included. This study therefore sought to accurately identify mosquito species from the three most epidemiologically important mosquito genera using wing morphometrics. Twelve mosquito species from three epidemiologically important genera (Aedes, Anopheles and Culex) were collected and identified by taxonomic keys. Next, the right wing of each adult female mosquito was removed and photographed, and the coordinates of eighteen digitized landmarks at the intersections of wing veins were collected. The allometric influence was assessed, and canonical variate analysis and thin-plate splines were used for species identification. Cross-validated reclassification tests were performed for each individual, and a Neighbor Joining tree was constructed to illustrate species segregation patterns. The analyses were carried out and the graphs plotted with TpsUtil 1.29, TpsRelw 1.39, MorphoJ 1.02 and Past 2.17c. Canonical variate analysis for Aedes, Anopheles and Culex genera showed three clear clusters in morphospace, correctly distinguishing the three mosquito genera, and pairwise cross-validated reclassification resulted in at least 99% accuracy; subgenera were also identified correctly with a mean accuracy of 96%, and in 88 of the 132 possible comparisons, species were identified with 100% accuracy after the data was subjected to reclassification. Our results showed that Aedes, Culex and Anopheles were correctly distinguished by wing shape. For the lower hierarchical levels (subgenera and species), wing geometric morphometrics was also efficient, resulting in high reclassification scores.

This parentage verification study was carried out on 14 full-sib families of Eucalyptus species. Verification of the correct paternity and maternity is a key step in accurately estimating quantitative genetic parameters as well as efficiently genotyping for genetic linkage map construction and phenotype assessment. Six microsatellite markers were used for this certification. Out of 305 progeny individuals, 78.03% were correctly assigned to the putative mother/father trees. Variable levels of contamination were detected in 10 of the 14 families. Alternative pollen parents could be identified among the parent trees tested for 4.5% of the offspring, revealing possible mislabeling in controlled crosses. The identification of several participating pollen parents in some families suggests pollen and/or seed mixture. This study highlights that the current controlled pollination methods used in Eucalyptus are subject to errors. Parentage certification should be used on a routine basis in controlled cross...

O Estado de São Paulo apresenta uma das mais altas taxas de infecções por vírus dengue no Mundo, mas apesar dessa situação, poucos são os estudos dirigidos às populações do mosquito Aedes aegypti. O objetivo deste trabalho foi caracterizar geneticamente e morfologicamente populações de Ae. aegypti localizadas em seis municípios (Santos, S.P., Campinas, São Carlos, Catanduva, S.J.R.P.) do Estado de São Paulo durante 2011 e 2012. Todos os marcadores biológicos indicaram estruturação populacional. Os oito loci microssatélites apontaram diferenciação genética moderada entre as populações (Fst= 0.04; p < 0,05) e os níveis de diversidade nucleotídica do gene COI (π =0,0062) e do gene ND4 (π=0,017) foram moderadamente altos. Duas linhagens geneticamente distintas foram encontradas no Estado. Ao longo dos meses que compreenderam o estudo, foram encontradas diferenças morfo-genéticas temporais entre as seis populações analisadas, possivelmente indicativas de microevolução. Os resultados obtidos podem ser úteis para compreendermos a dispersão deste mosquito vetor. The State of São Paulo displays one of the highest rates of dengue infection in the world, but despite this fact, a few populational studies of Ae. aegypti have been undertaken. The aim of this study was to genetically and morphologically characterize Ae. aegypti populations from six locations in the São Paulo State (Santos, S.P., Campinas, São Carlos, Catanduva, S.J.R.P.) during 2011 and 2012. The phenetic and genetic analyses revealed that populations of Ae. aegypti are structured. Eight microsatellites loci were polymorphic and genetic differentiation among samples was moderate (Fst= 0.04; p < 0.05). Nucleotide diversities of COI (π = 0.0062) and ND4 gene (π = 0.017) were moderately high. Two lineages distinct genetically were found in the State. Over the months comprised by the study, we found the temporal genetics and morphologics differences among the six populations, a possibly indicative of microevolution of mosquitoes. The results of this study may be useful for understand the spread of this vector mosquitoes in the State of São Paulo.

Background Sexual dimorphism in animals has been studied from different perspectives for decades. In 1874 Darwin hypothesized that it was related to sexual selection, and even after nearly 140 years, when additional empirical data has become available and the subject has been investigated from a contemporary viewpoint, this idea is still supported. Although mosquito (Culicidae) wings are of great importance as they play a sex-specific role, little is known about wing sexual dimorphism in these pathogen-vector insects. Detection and characterization of wing sexual dimorphism in culicids may indirectly enhance our knowledge of their epidemiology or reveal sex-linked genes, aspects that have been discussed by vector control initiatives and developers of genetically modified mosquitoes. Methods Using geometric morphometrics, we carried out a comparative assessment of wing sexual dimorphism in ten culicid species of medical/veterinary importance from genera Culex, Aedes, Anopheles and Ochlerotatus collected in Brazil. Results Discriminant analysis revealed significant sexual dimorphism in all the species studied, indicating that phenotypic expression of wing shape in mosquitoes is indeed sex-specific. A cross-validated test performed to reclassify the sexes with and without allometry yielded very similar results. Mahalanobis distances among the ten species showed that the species had different patterns of shape sexual dimorphism and that females are larger than males in some species. Conclusion Wing morphology differed significantly between species. The finding of sexual dimorphism in all the species would suggest that the wing geometry of Culicidae is canalized. Although sexual dimorphism is prevalent, species-specific patterns occur. Allometry was not the main determinant of sexual dimorphism, which suggests that sexual selection or other evolutionary mechanisms underlie wing sexual dimorphism in these insects.

Aedes aegypti is the most important vector of dengue viruses in tropical and subtropical regions. Because vaccines are still under development, dengue prevention depends primarily on vector control. Population genetics is a common approach in research involving Ae. aegypti. In the context of medical entomology, wing morphometric analysis has been proposed as a strong and low-cost complementary tool for investigating population structure. Therefore, we comparatively evaluated the genetic and phenotypic variability of population samples of Ae. aegypti from four sampling sites in the metropolitan area of São Paulo city, Brazil. The distances between the sites ranged from 7.1 to 50km. This area, where knowledge on the population genetics of this mosquito is incipient, was chosen due to the thousands of dengue cases registered yearly. The analysed loci were polymorphic, and they revealed population structure (global FST=0.062; p

Background Culex quinquefasciatus mosquitoes can be found in almost every major city of Brazil and are vectors of filariasis and several arboviruses. Microsatellite markers have been widely used to uncover the genetic structure of various groups of insect populations. The aim of this study was to glimpse the genetic structure of Cx. quinquefasciatus in Brazil. Methods Nine populations were sampled across Brazil (one of them from a laboratory colony - COL) and another one from Argentina and process regarding the variability of six microsatellite loci. Results The analyzed loci revealed moderate population genetic structure (mean Fst = 0.12). Dendrograms of genetic distances evidenced two major population clusters, respectively corresponding to the northern and southern populations. The hybrid population Cx. pipiens/quinquefasciatus (from La Plata, Argentina) and the colony population fell outside the major clusters. Those clusters were substructured and there was a significant correlation between genetic and geographic distances and environmental variables (r = 0.51; p > 0.001 and r = 0.46; p > 0.004). Conclusions Multilocus cluster Bayesian analysis confirmed that populations are mutually distinct, and the set of results point to genetic differences among populations. The presumable low gene flow among them may be due to the large geographic distances (>1000 km) and to the environmental heterogeneity of the sampled areas. The genetic structure observed in this study may lead to the best understanding of Cx. quinquefasciatus demographical diversity as well as their genetic variations patterns in Brazil so far unknown.