SINE-B1 Distribution and Chromosome Rearrangements in the South American Proechimys gr. goeldii (Echimyidae, Rodentia).

Proechimys species are remarkable for their extensive chromosome rearrangements, representing a good model to understand genome evolution. Herein, we cytogenetically analyzed 3 different cytotypes of Proechimys gr. goeldii to assess their evolutionary relationship. We also mapped the transposable element SINE-B1 on the chromosomes of P. gr. goeldii in order to investigate its distribution among individuals and evaluate its possible contribution to karyotype remodeling in this species. SINE-B1 showed a dispersed distribution along chromosome arms and was also detected at the pericentromeric regions of some chromosomes, including pair 1 and the sex chromosomes, which are involved in chromosome rearrangements. In addition, we describe a new cytotype for P. gr. goeldii, reinforcing the significant role of gross chromosomal rearrangements during the evolution of the genus. The results of FISH with SINE-B1 suggest that this issue should be more deeply investigated for a better understanding of its role in the mechanisms involved in the wide variety of Proechimys karyotypes.

Identification of bacterial infection in neotropical primates.

Emerging infectious diseases usually arise from wild animal populations. In the present work, we performed a screening for bacterial infection in natural populations of New World primates. The blood cell bulk DNAs from 181 individuals of four Platyrrhini genera were PCR screened for eubacterial 16S rRNA genes. Bacteria were detected and identified in 13 distinct individuals of Alouatta belzebul, Alouatta caraya, and Cebus apella monkeys from geographically distant regions in the states of Mato Grosso and Pará, Brazil. Sequence analyses showed that these Platyrrhini bacteria are closely related not only to human pathogens Pseudomonas spp. but also to Pseudomonas simiae and sheep-Acari infecting Pseudomonas spp. The identified Pseudomonas possibly represents a group of bacteria circulating in natural monkey populations.

Molecular epidemiology of Laguna Negra virus, Mato Grosso State, Brazil.

We associated Laguna Negra virus with hantavirus pulmonary syndrome in Mato Grosso State, Brazil, and a previously unidentified potential host, the Calomys callidus rodent. Genetic testing revealed homologous sequencing in specimens from 20 humans and 8 mice. Further epidemiologic studies may lead to control of HPS in Mato Grosso State.

Detection of the first incidence of Akodon paranaensis naturally infected with the Jabora virus strain (Hantavirus) in Brazil.

We characterised hantaviruses circulating in different Akodon rodent species collected in midwestern Santa Catarina (SC), southern Brazil, where the Jabora hantavirus (JABV) strain was first identified in Akodon montensis. Genetic and phylogenetic analyses based on a partial S segment indicated that, in SC, Akodon paranaensis and A. montensis carried the same type of hantavirus. Additionally, we conducted the first genomic characterisation of the complete S segment from the Brazilian JABV strain. This is the first report of A. paranaensis infected with the JABV.

Karyotypical Confirmation of Natural Hybridization between Two Manatee Species, Trichechus manatus and Trichechus inunguis.

Two species of manatees are found in Northern Brazil-the Antillean manatee (Trichechus manatus), which is found along the coast from Florida to Northeastern Brazil, and the Amazonian manatee (Trichechus inunguis), endemic to the Amazon drainage basin. These species show a sympatric distribution in the region of the Marajó Archipelago, an estuarine area surrounding the Amazon River mouth. There is evidence of the occurrence of interspecific hybrids in this area, based on mitochondrial DNA analyses, although the use of nuclear markers has not corroborated this proposal. Considering that these species show very distinct karyotypes, despite being closely related (2n = 48 in T. manatus and 2n = 56 in T. inunguis), hybrids would present distinct chromosome numbers. Based on this, we conducted cytogenetic analyses using classic and molecular techniques in three calves found stranded in the Marajó Island and Amapá coast. The results showed that one of them, morphologically classified as T. inunguis, presented the correspondent karyotype, with 2n = 56. However, the other two, which were phenotypically similar to T. manatus, showed 2n = 49. Despite the same diploid number, their G-banding patterns revealed some differences. The results of the distribution of some microsatellite sequences have also confirmed the heterozygosity of some chromosomal pairs in these two individuals. These results are the first indubitable confirmation of the occurrence of natural hybrids between T. manatus and T. inunguis, and also brings about some issues concerning the viability of hybrids, considering that these two individuals do not correspond to an F1 hybrid, but instead, both presented a possible F2 karyotype.

Pygmy rice rat as potential host of Castelo dos Sonhos Hantavirus.

To study the dynamics of wild rodent populations and identify potential hosts for hantavirus, we conducted an eco-epidemiologic study in Campo Novo do Parecis, Mato Grosso State, Brazil. We detected and genetically characterized Castelo dos Sonhos virus found in a species of pygmy rice rat (Oligoryzomys utiaritensis).

Clonal analysis of hepatitis B viruses among blood donors from Joinville, Brazil: evidence of dual infections, intragenotype recombination and markers of risk for hepatocellular carcinoma.

Hepatitis B virus (HBV) is classified into seven major genotypes (A-H). Brazil, a country of continental proportions, has three prevailing lineages of HBV genotypes A, D, and F. Distinct HBV genotypes have been associated with differential risk of disease progression. Pre-S gene deletions and single nucleotide polymorphisms have also been linked to progression to liver diseases. In this study, the molecular epidemiology of HBV was examined in Southern Brazil. The occurrence of multiple HBV infections, HBV recombination, and genetic markers of disease progression were also evaluated. Seventy-eight persons infected with HBV had their viruses characterized molecularly by nested PCR, DNA sequencing, and phylogenetic inference. Multiple infections and recombinant viruses were evaluated by clonal and bootscanning analyses. The vast majority (96%) of the strains belonged to different D subgenotypes. Three of the four strains with unresolved genotypic classification showed evidence of dual infections with distinct D subgenotypes by clonal analysis. There was also evidence of intragenotype mosaic viruses. While four viruses had pre-S deletions as major variants, another two displayed minor variants with such characteristics. One strain carried the F141L mutation, associated recently with increased risk of hepatocellular carcinoma. These results emphasize the need for monitoring HBV genotype distribution around South America, as well as for the presence of genetic markers of disease progression in subjects diagnosed with HBV recently.

Human Papillomavirus in Brazilian women with and without cervical lesions.

BACKGROUND: Human Papillomavirus (HPV) high-risk (HR) types are the causal factor for cervical cancer and premalignant dysplasia. Data on frequency of HPV types provide a basis to design and evaluate HPV prevention programs. Taking into account the heterogeneity of HPV types across and within populations this study aims to access the HPV frequency in Brazilian women. RESULTS: We identified 24 different types of HPV, including a Betapapillomavirus and a likely new type, previously reported, from 132 women positive for the virus analysed by Hybrid Capture II assay. These women were infected by a single or multiple HPV types and 142 HPV strains were identified. HR types were found in 75% of women and HPV types 16, 18, 45, 58, and 66 had the highest frequency. Significant differences in frequency of HR HPV types were found for presence of cervical lesions, and for different HPV species and women age. CONCLUSIONS: Compared with previous studies in Brazil, our data indicated differences in frequency and HPV type diversity, a significant association of other HR-types but HPV16 and 18 and cervical lesions, and a trend for distinct distribution of HPV types by age.

Colonization process of the Brazilian common vesper mouse, Calomys expulsus (Cricetidae, Sigmodontinae): a biogeographic hypothesis.

Riverine barriers have been associated to genetic diversification and speciation of several taxa. The Rio São Francisco is one of the largest rivers in South America, representing the third largest river basin in Brazil and operating as a geographic barrier to gene flow of different taxa. To evaluate the influence of the Rio São Francisco in the speciation of small rodents, we investigated the genetic structure of Calomys expulsus with phylogenetic and network analyses of cytochrome b DNA. Our results suggested that C. expulsus can be divided into 3 subpopulations, 2 on the left and another one on the right bank of this river. The time of divergence of these subpopulations, using a Bayesian framework, suggested colonization from the south to the north/northeast. Spatial analysis using a clustering method and the Monmonier's algorithm suggested that the Rio São Francisco is a biogeographic barrier to gene flow and indicated that this river may play a role in the incipient speciation process of these subpopulations.

Identification, classification and evolution of owl monkeys (Aotus, Illiger 1811).

BACKGROUND: Owl monkeys, belonging to the genus Aotus, have been extensively used as animal models in biomedical research but few reports have focused on the taxonomy and phylogeography of this genus. Moreover, the morphological similarity of several Aotus species has led to frequent misidentifications, mainly at the boundaries of their distribution. In this study, sequence data from five mitochondrial regions and the nuclear, Y-linked, SRY gene were used for species identification and phylogenetic reconstructions using well characterized specimens of Aotus nancymaae, A. vociferans, A. lemurinus, A. griseimembra, A. trivirgatus, A. nigriceps, A. azarae boliviensis and A. infulatus. RESULTS: The complete MT-CO1, MT-TS1, MT-TD, MT-CO2, MT-CYB regions were sequenced in 18 Aotus specimens. ML and Bayesian topologies of concatenated data and separate regions allowed for the proposition of a tentative Aotus phylogeny, indicating that Aotus diverged some 4.62 Million years before present (MYBP). Similar analyses with included GenBank specimens were useful for assessing species identification of deposited data. CONCLUSIONS: Alternative phylogenetic reconstructions, when compared with karyotypic and biogeographic data, led to the proposition of evolutionary scenarios questioning the conventional diversification of this genus in monophyletic groups with grey and red necks. Moreover, genetic distance estimates and haplotypic differences were useful for species validations.

Hantaviruses and hantavirus pulmonary syndrome, Maranhao, Brazil.

To confirm circulation of Anajatuba virus in Maranhao, Brazil, we conducted a serologic survey (immunoglobulin G ELISA) and phylogenetic studies (nucleocapsid gene sequences) of hantaviruses from wild rodents and persons with hantavirus pulmonary syndrome. This virus is transmitted by Oligoryzomys fornesi rodents and is responsible for hantavirus pulmonary syndrome in this region.

Mitochondrial divergence between 2 populations of the hooded capuchin, Cebus (Sapajus) cay (Platyrrhini, Primates).

We analyzed the molecular divergence of 2 separate populations of Cebus apella paraguayanus, recently considered a junior synonym of Cebus cay, and estimated its time of separation from C. apella. Cytochrome b DNA from 23 C. cay from Brazil and 9 from Paraguay showed 24 haplotypes (20 and 4, respectively), accounting for 29 variable sites (19 transitions and 10 transversions), with 40.0%, 26.7%, and 33.0% replacements at first, second, and third codon positions, respectively. Genetic distance between haplotypes averaged 0.5%, with 1.1% between C. cay populations. Phylogenetic reconstructions and median joining separated C. cay from Brazil and Paraguay. Neighbor joining showed C. cay and C. apella as sister groups, although C. cay and C. apella collapsed in maximum parsimony and maximum likelihood topologies. Analysis of molecular variance showed the highest variance component between C. cay populations, and mismatch distribution indicated that this species suffered a recent demographic expansion. Divergence time estimates suggested that the 2 populations of C. cay split in the Pleistocene, a period of repeated glaciation events leading to drastic changes in the vegetation composition of different biomes.

Morphological variation in the genus Juliomys (Rodentia: Cricetidae: Sigmodontinae) and taxonomic status of Juliomys anoblepas (Winge 1887) from the Quaternary of Southeast Brazil.

The region of Lagoa Santa, Minas Gerais, Brazil, is one of the most important karstic areas of the Brazilian Quaternary due to the faunistic diversity of living and extinct forms. Among them, some taxa remain poorly studied, as is the case of Calomys anoblepas Winge 1887. Despite the recent allocation of the taxon within Juliomys, its description and morphological analysis are condensed, based on comparative few specimens and on few informative characters. In this study, we investigate characters proposed to distinguish species of Juliomys, and reevaluate the taxonomic status of the fossil Juliomys anoblepas. We analyzed 80 cranio-dental morphological characters in 233 specimens represented by the four species currently recognized: J. pictipes (Osgood 1933), J. rimofrons Oliveira Bonvicino 2002, J. ossitenuis Costa, Pavan, Leite Fagundes 2007, and J. ximenezi Christoff, Vieira, Oliveira, Gonçalves, Valiati Tomasi 2016. We also performed principal component analysis on eight craniodental measurements available for the J. anoblepas hypodigm. The review of morphological systems and the evaluation of the characters used in the literature revealed that there are no diagnostic characters in the anterior portion of the skull and in the molar series of Juliomys, being difficult to differentiate the fossil from the other living species. Only six qualitative characters were variable and applicable to the hypodigm of J. anoblepas. Characters are polymorphic, invariable, or the fossil is not sufficiently complete to determinate its states. The taxon could not be morphometrically differentiated from J. pictipes and J. ossitenuis. Based on the results presented herein, we consider J. anoblepas as a nomen dubium and restrict its name to the taxon's hypodigm.

Constitutive RB1 mutation in a child conceived by in vitro fertilization: implications for genetic counseling.

BACKGROUND: The purpose of this study was to identify mutations associated with bilateral retinoblastoma in a quadruplet conceived by in vitro fertilization, and to trace the parental origin of mutations in the four quadruplets and their father. METHODS: Mutational screening was carried out by sequencing. Genotyping was carried out for determining quadruplet zygosity. RESULTS: The proband was a carrier of a novel RB1 constitutive mutation (g.2056C>G) which was not detected in her father or her unaffected sisters, and of two other mutations (g.39606 C>T and g.174351T>A) also present in two monozygotic sisters. The novel mutation probably occurred de novo while the others were of likely maternal origin. The novel mutation, affecting the Kozak consensus at the 5'UTR of RB1 and g.174351T>A were likely associated to retinoblastoma in the proband. CONCLUSION: Molecular diagnosis of retinoblastoma requires genotypic data of the family for determining hereditary transmission. In the case of children generated by IVF with oocytes from an anonymous donor which had been stored in a cell repository, this might not be successfully accomplished, making precise diagnosis impracticable for genetic counseling.

Divergence in Zygodontomys (rodentia: Sigmodontinae) and distribution of amazonian savannas.

Northern South America presents a diverse array of nonforest or savanna-like ecosystems that are patchily distributed. The distribution of these open habitats has been quite dynamic during Quaternary glacial-interglacial cycles; yet, the relevance of climatically driven vicariance events to the diversification of nonforest Amazonian vertebrates remains poorly known. We analyzed karyologic and mitochondrial DNA sequence data of the genus Zygodontomys, a small cricetid rodent distributed throughout nonforest habitats of northern Amazonia. Samples analyzed represented 4 Brazilian Amazonian localities and 2 French Guiana localities. Karyologic variation among Amazonian Brazilian Zygodontomys populations is high, with, at least, 3 karyomorphotypes. Molecular phylogenetic analyses recovered 3 major clades congruent with known karyotypes, a finding that suggests the existence of 3 species, 2 of which currently undescribed. The French Guiana and Surumú clade, identified as Zygodontomys brevicauda microtinus, is characterized by 2n = 86 and is sister to the clade formed by the 2 nondescribed forms. The Rio Negro-Rio Branco form is characterized by 2n = 82, and the Ferreira Gomes-Itapoá form is characterized by 2n = 84. The distribution of the 3 Zygodontomys lineages identified is in accordance with the geography of the open vegetation patches in Northern Amazonia, and divergence time estimates relate speciation events to the middle-upper Pleistocene, supporting the prominent role of Quaternary climatically driven vicariance events in the diversification of the genus.

Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies.

IMPORTANCE: Non-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined. OBJECTIVES: The aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches. DESIGN SETTINGS AND PARTICIPANTS: Ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies (4 babies with maternal ZIKV exposure during gestation and presence of clinical signs which included ocular abnormalities and microcephaly; 4 babies with maternal ZIKV exposure during gestation but no clinical signs; and 4 unaffected control babies with negative PCR for Zika virus and without clinical signs). Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments. RESULTS: Our optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations (cell keratinization, pyknosis, karyolysis, anucleation, and vacuolization). Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies. CONCLUSIONS AND RELEVANCE: Our method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research.

DNA barcoding of the rodent genus Oligoryzomys (Cricetidae: Sigmodontinae): mitogenomic-anchored database and identification of nuclear mitochondrial translocations (Numts).

DNA barcoding has become a standard method for species identification in taxonomically complex groups. An important step of the barcoding process is the construction of a library of voucher-based material that was properly identified by independent methods, free of inaccurate identification, and paralogs. We provide here a cytochrome oxidase I (mt-Co1) DNA barcode database for species of the genus Oligoryzomys, based on type material and karyotyped specimens, and anchored on the mitochondrial genome of one species of Oligoryzomys, O. stramineus. To evaluate the taxonomic determination of new COI sequences, we assessed species intra/interspecific genetic distances (barcode gap), performed the General Mixed Yule Coalescent method (GMYC) for lineages' delimitation, and identified diagnostic nucleotides for each species of Oligoryzomys. Phylogenetic analyses of Oligoryzomys were performed on 2 datasets including 14 of the 23 recognized species of this genus: a mt-Co1 only matrix, and a concatenated matrix including mt-Co1, cytochrome b (mt-Cytb), and intron 7 of the nuclear fibrinogen beta chain gene (i7Fgb). We recovered nuclear-mitochondrial translocated (Numts) pseudogenes on our samples and identified several published sequences that are cases of Numts. We analyzed the rate of non-synonymous and synonymous substitution, which were higher in Numts in comparison to mtDNA sequences. GMYC delimitations and DNA barcode gap results highlight the need for further work that integrate molecular, karyotypic, and morphological analyses, as well as additional sampling, to tackle persistent problems in the taxonomy of Oligoryzomys.

Hereditary retinoblastoma transmitted by maternal germline mosaicism.

BACKGROUND: Investigating transmission of a constitutive, g78238C > T (R552X), RB1 mutation in four affected children descended from three different unaffected fathers and an unaffected mother. PROCEDURES: Sequence data analyses and allele-specific PCR assays were used to investigate the presence of the mutation in four affected children, five unaffected sibs (or half-sibs), and the unaffected mother. Haplotyping was carried out for confirming that the children descended from different fathers. RESULTS: Haplotyping excluded the possibility of paternal transmission of a de novo mutation and provided evidence of maternal germline mosaicism. The mutation was apparently absent in blood- and buccal cell-DNA of the mother who also showed a normal fundoscopy. CONCLUSIONS: Our findings indicated that mosaicism was restricted to the maternal germline. The mutational event must have occurred at least 4 weeks post-conception, unlike the early mutational events of most mosaics, occurring between fertilization and the 8th day of conception. The implications of these findings are discussed in view that genetic counselling should discriminate between germline mosaicism and de novo events in pseudo-low-penetrant hereditary retinoblastoma.

Integrative taxonomy of the Amazonian red-sided opossum Monodelphis glirina (J.A. Wagner, 1842) (Didelphimorphia: Didelphidae).

New records for Monodelphis glirina, a short-tailed opossum distributed throughout the Amazon region, from Humaitá, Amazonas state, and Confresa, Mato Grosso state, prompted new insights into the geographic distribution and genetic diversity of this species. One of the records extends the species range circa 350 km beyond the previous southeastern limit. Voucher specimens had their identification confirmed by morphological comparison with the holotype and corroborated by molecular data (mitochondrial gene Cytochrome b). Our analyses revealed an east-west geographic structure into two clades that could represent distinct species, M. glirina and M. maraxina. Furthermore, examination of the holotype brought to light that it was never formally linked with the voucher specimen collected by Johann Natterer.