Carolina M. Voloch

Congenital Zika Virus Infection: Beyond Neonatal Microcephaly

Importance Recent studies have reported an increase in the number of fetuses and neonates with microcephaly whose mothers were infected with the Zika virus (ZIKV) during pregnancy. To our knowledge, most reports to date have focused on select aspects of the maternal or fetal infection and fetal effects. Objective To describe the prenatal evolution and perinatal outcomes of 11 neonates who had developmental abnormalities and neurological damage associated with ZIKV infection in Brazil. Design, Setting, and Participants We observed 11 infants with congenital ZIKV infection from gestation to 6 months in the state of Paraíba, Brazil. Ten of 11 women included in this study presented with symptoms of ZIKV infection during the first half of pregnancy, and all 11 had laboratory evidence of the infection in several tissues by serology or polymerase chain reaction. Brain damage was confirmed through intrauterine ultrasonography and was complemented by magnetic resonance imaging. Histopathological analysis was performed on the placenta and brain tissue from infants who died. The ZIKV genome was investigated in several tissues and sequenced for further phylogenetic analysis. Main Outcomes and Measures Description of the major lesions caused by ZIKV congenital infection. Results Of the 11 infants, 7 (63.6%) were female, and the median (SD) maternal age at delivery was 25 (6) years. Three of 11 neonates died, giving a perinatal mortality rate of 27.3%. The median (SD) cephalic perimeter at birth was 31 (3) cm, a value lower than the limit to consider a microcephaly case. In all patients, neurological impairments were identified, including microcephaly, a reduction in cerebral volume, ventriculomegaly, cerebellar hypoplasia, lissencephaly with hydrocephalus, and fetal akinesia deformation sequence (ie, arthrogryposis). Results of limited testing for other causes of microcephaly, such as genetic disorders and viral and bacterial infections, were negative, and the ZIKV genome was found in both maternal and neonatal tissues (eg, amniotic fluid, cord blood, placenta, and brain). Phylogenetic analyses showed an intrahost virus variation with some polymorphisms in envelope genes associated with different tissues. Conclusions and Relevance Combined findings from clinical, laboratory, imaging, and pathological examinations provided a more complete picture of the severe damage and developmental abnormalities caused by ZIKV infection than has been previously reported. The term congenital Zika syndrome is preferable to refer to these cases, as microcephaly is just one of the clinical signs of this congenital malformation disorder.

Comparative evolutionary epidemiology of dengue virus serotypes.

Evolutionary studies on dengue virus have frequently focused on intra-serotype diversity or on specific epidemics. In this study, we compiled a comprehensive data set of the envelope gene of dengue virus serotypes and conducted an extensive comparative study of evolutionary molecular epidemiology. We found that substitution rates are homogeneous among dengue serotypes, although their population dynamics have differed over the past few years as inferred by Bayesian coalescent methods. On a global scale, DENV-2 is the serotype with the highest effective population size. The genealogies also showed geographical structure within the serotypes. Finally, we also explored the causes of dengue virus serotype diversification by investigating the plausibility that it was driven by adaptive changes. Our results suggest that the envelope gene is under significant purifying selection and the hypothesis that dengue virus serotype diversification was the result of stochastic events cannot be ruled out.

Genetic structure of the sea-bob shrimp (Xiphopenaeus kroyeri Heller, 1862; Decapoda, Penaeidae) along the Brazilian southeastern coast

The sea-bob shrimp, Xiphopenaeus kroyeri, is one of the most important economic marine resources along the entire Brazilian coast. Nevertheless, despite its economic importance, no studies have examined the population genetics of this species. In this paper, we used ten allozyme loci to study the pattern of genetic structuring in X. kroyeri along the southeastern Brazilian coast. Seven of the ten analyzed loci were polymorphic, yielding observed heterozygosity values higher than those reported for other penaeid shrimps. The population from Sao Paulo was significantly different from the other two populations (Rio de Janeiro and Espirito Santo), which, in turn, seem to form a single panmitic unit. Therefore, our results clearly indicate that conservation policies for this species should consider the Sao Paulo population as an independent stock from those of Rio de Janeiro and Espirito Santo.

Diversification of the Genus Anopheles and a Neotropical Clade from the Late Cretaceous

The Anopheles genus is a member of the Culicidae family and consists of approximately 460 recognized species. The genus is composed of 7 subgenera with diverse geographical distributions. Despite its huge medical importance, a consensus has not been reached on the phylogenetic relationships among Anopheles subgenera. We assembled a comprehensive dataset comprising the COI, COII and 5.8S rRNA genes and used maximum likelihood and Bayesian inference to estimate the phylogeny and divergence times of six out of the seven Anopheles subgenera. Our analysis reveals a monophyletic group composed of the three exclusively Neotropical subgenera, Stethomyia, Kerteszia and Nyssorhynchus, which began to diversify in the Late Cretaceous, at approximately 90 Ma. The inferred age of the last common ancestor of the Anopheles genus was ca. 110 Ma. The monophyly of all Anopheles subgenera was supported, although we failed to recover a significant level of statistical support for the monophyly of the Anopheles genus. The ages of the last common ancestors of the Neotropical clade and the Anopheles and Cellia subgenera were inferred to be at the Late Cretaceous (ca. 90 Ma). Our analysis failed to statistically support the monophyly of the Anopheles genus because of an unresolved polytomy between Bironella and A. squamifemur.

Impact of the Partitioning Scheme on Divergence Times Inferred from Mammalian Genomic Data Sets

Data partitioning has long been regarded as an important parameter for phylogenetic inference. The division of heterogeneous multigene data sets into partitions with similar substitution patterns is known to increase the performance of probabilistic phylogenetic methods. However, the effect of the partitioning scheme on divergence time estimates has generally been ignored. To investigate the impact of data partitioning on the estimation of divergence times, we have constructed two genomic data sets. The first one with 15 nuclear genes comprising 50,928 bp were selected from the OrthoMam database; the second set was composed of complete mitochondrial genomes. We studied two partitioning schemes: concatenated supermatrices and partitioned gene analysis. We have also measured the impact of taxonomic sampling on the estimates. After drawing divergence time inferences using the uncorrelated relaxed clock in BEAST, we have compared the age estimates between the partitioning schemes. Our results show that, in general, both schemes resulted in similar chronological estimates, however the concatenated data sets were more efficient than the partitioned ones in attaining suitable effective sample sizes.

Amblyomma sculptum: genetic diversity and rickettsias in the Brazilian Cerrado biome

Amblyomma sculptum (Ixodida: Ixodidae) Berlese, 1888 is the most important tick vector in Brazil, transmitting the bioagent of the most severe form of spotted fever (SF) in part of the Cerrado (in the states of Minas Gerais and São Paulo). In another part of the Cerrado (Central‐West region of Brazil), a milder form of SF has been recorded. However, neither the rickettsia nor the vector involved have been characterized. The aim of the current study was to analyse genetic variation and the presence of rickettsia in A. sculptum in Cerrado, from silent areas and with the milder form of SF. Samples were subjected to DNA extraction, amplification and sequencing of 12S rDNA, cytochrome oxidase subunit II and D‐loop mitochondrial genes (for tick population analyses), and gltA, htrA, ompA and gene D (sca4) genes for rickettsia researches. Exclusive haplotypes with low frequencies, high haplotype diversity and low nucleotide diversity, star‐shaped networks and significant results in neutrality tests indicate A. sculptum population expansions in some areas. Rickettsia amblyommatis, Candidatus Rickettsia andeanae and Rickettsia felis were detected. The A. sculptum diversity is not geographically, or biome delimited, pointing to a different potential in vector capacity, possibly associated with differing tick genetic profiles.

Analysis of Amblyomma sculptum haplotypes in an area endemic for Brazilian spotted fever

Amblyomma sculptum (Ixodida: Ixodidae) Berlese, 1888, a member of the Amblyomma cajennense complex, is the major vector of Brazilian spotted fever (BSF) in southeastern Brazil. In this study, the genetic diversity of A. sculptum populations in the state of Rio de Janeiro (RJ), Brazil, was investigated because genetic variability in tick populations may be related to vector competence. Samples of A. sculptum from 19 municipalities in 7 regions of RJ were subjected to DNA extraction, amplification and sequencing of D‐loop, cytochrome oxidase II and 12S rDNA mitochondrial genes. These sequences were used to map the genetic diversity of this tick. Amblyomma sculptum populations are genetically diverse in RJ, especially in the South Centre and Highland regions. Few unique haplotypes were observed in all populations, and the majority of genetic variation found was among ticks within each population. Phylogenetic reconstruction reinforced the assumption that all the haplotypes identified in RJ belong to A. sculptum. However, some RJ haplotypes are closer to A. sculptum from Argentina than to A. sculptum from elsewhere in Brazil. In RJ, A. sculptum has high genetic diversity, although little genetic differentiation. Observations also indicated a high level of gene flow among the studied populations and no evidence of population structure according to region in RJ.

Analysis of adaptive evolution in Lyssavirus genomes reveals pervasive diversifying selection during species diversification.

Lyssavirus is a diverse genus of viruses that infect a variety of mammalian hosts, typically causing encephalitis. The evolution of this lineage, particularly the rabies virus, has been a focus of research because of the extensive occurrence of cross-species transmission, and the distinctive geographical patterns present throughout the diversification of these viruses. Although numerous studies have examined pattern-related questions concerning Lyssavirus evolution, analyses of the evolutionary processes acting on Lyssavirus diversification are scarce. To clarify the relevance of positive natural selection in Lyssavirus diversification, we conducted a comprehensive scan for episodic diversifying selection across all lineages and codon sites of the five coding regions in lyssavirus genomes. Although the genomes of these viruses are generally conserved, the glycoprotein (G), RNA-dependent RNA polymerase (L) and polymerase (P) genes were frequently targets of adaptive evolution during the diversification of the genus. Adaptive evolution is particularly manifest in the glycoprotein gene, which was inferred to have experienced the highest density of positively selected codon sites along branches. Substitutions in the L gene were found to be associated with the early diversification of phylogroups. A comparison between the number of positively selected sites inferred along the branches of RABV population branches and Lyssavirus intespecies branches suggested that the occurrence of positive selection was similar on the five coding regions of the genome in both groups.

Beads and Dice in a Genetic Drift Exercise

Natural selection driving adaptive changes is a powerful and intuitive explanation for the evolution of the living world around us. Evolution at the molecular level, however, is chiefly ruled by random genetic drift. The idea that an advantageous allele may be lost by chance in a natural population is rather difficult to explore in the classroom. Low-cost and hands-on educational resources are needed to make genetic drift more intuitive among students. In this exercise, we use colored beads and the roll of a die to simulate drift and selection jointly affecting the fate of the genetic variants in an evolving population. Our aim is to teach students that natural selection does not determine but simply influences the fate of advantageous alleles because random genetic drift is always present. We have been using this exercise successfully for over a decade for the Biological Sciences students at the Federal University of Rio de Janeiro.

Ultrastructural Morphology and Molecular Analyses of Tropical and Temperate "Species" of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) in Brazil

Abstract The Rhipicephalus sanguineus (Latreille) complex (Acari:Ixodidae) is composed of species with intra- and interspecific morphological variation that make their diagnosis difficult. In the present study, male specimens of the R. sanguineus complex were collected from dogs in six districts of three regions of Brazil and submitted to molecular and scanning electron microscopy (SEM) analyses. Analysis of COX1 gene, 12S rDNA, and D-loop rDNA shows that ticks classified as R. sanguineus form two different clades. Morphological comparisons using SEM found adult males to exhibit morphological differences in Hallers organ, festoons, and adanal, spiracular, and genital plates, with the last having potential usefulness in distinguishing male specimens of the complex.