Hantavirus pulmonary syndrome and rodent reservoirs in the savanna-like biome of Brazil's southeastern region.

This paper describes the diversity of rodent fauna in an area endemic for hantavirus cardiopulmonary syndrome (HCPS) in Brazil, the population dynamics and the relationship of rodents with hantavirus in the Cerrado (savanna-like) biome. Additionally, an analysis is made of the partial S segment sequences of the hantaviruses obtained from serologically confirmed human HCPS cases and from rodent specimens. Rodents were collected during four campaigns. Human serum samples were collected from suspected cases of HCPS at hospitals in the state of Minas Gerais. The samples antibody-reactive by ELISA were processed by RT-PCR. The PCR product was amplified and sequenced. Hantavirus was detected only in Necromys lasiurus, the wild rodent species most prevalent in the Cerrado biome (min-max: 50-83·7%). All the six human serum samples were hantavirus seropositive and five showed amplified PCR products. The analysis of the nucleotide sequences showed the circulation of a single genotype, the Araraquara hantavirus. The environmental changes that have occurred in the Cerrado biome in recent decades have favoured N. lasiurus in interspecific competition of habitats, thus increasing the risk of contact between humans and rodent species infected with hantavirus. Our data corroborate the definition of N. lasiurus as the main hantavirus reservoir in the Cerrado biome.

Genetic diversity of neotropical primates: phylogeny, population genetics, and animal models for infectious diseases.

The classification of neotropical primates has been controversial, and different arrangements have been proposed based on disparate taxonomic criteria and on the traits selected for elucidating phylogenetic reconstructions, like morphologic characters, nuclear DNA and mitochondrial DNA. Population studies of some neotropical primates have been useful for assessing their extant genetic variability and for understanding their social structure and dynamics. Finally, neotropical primates have become valuable models for some human infectious deseases, especially for HIV studies related to viral resistance. In this review, we comment on these aspects that make neotropical primates a group of highly valuable species for basic and applied research.

Population genetic studies of Alouatta belzebul from the Amazonian and Atlantic Forests.

Cytochrome b DNA sequence data (ca. 1,140 bp) of 66 Alouatta belzebul from the Amazonian and the Atlantic Forests of Brazil were used for phylogenetic reconstructions and population studies. Our sample consisted of 60 specimens from the Amazonian Forest (captured in 1984 and 1998 in Pará-PA state) and six specimens from the Atlantic Forest (Paraíba-PB state). We found 32 haplotypes, 23 in PA-1984 (with 12 present in more than one individual), 11 in PA-1998 (with two present in more than one individual), and a single haplotype in the PB sample. Animals from PA-1984 and PA-1998 shared three haplotypes while animals from Pará and Paraíba did not share any haplotype. We found 57 variable sites, consisting of 53 transitions and four transversions, with most replacements occurring at third codon position (77.19%) and less frequently at first and second positions (10.53 and 12.28%, respectively). Genetic distance between all haplotypes varied between 0 and 1.2%. Nucleotide diversity estimates between PA-1984 haplotypes and PA-1998 haplotypes were the same (π=0.01), and haplotype diversity estimates were very similar (h=0.96 and 0.93 for PA-1984 and PA-1998, respectively). Maximum parsimony, median-joining, split decomposition, and TCS showed that PA and PB haplotypes had not drastically diverged and that subsequent radiation within these regions was not apparent. No temporal structure was found between PA-1984 and PA-1998. The sum of square deviation estimate for PA-1984 equaled 0.01 (P=0.23), in agreement with a hypothetical model of sudden expansion contrary to PA-1998 whose sum of square deviation estimate (0.40; P=0.04) was not compatible with this model, although the small sample size of PA-1998 as well as the smaller area of capture could have also accounted for this result. Fu's F(s) and R(2) statistical neutrality tests corroborated these propositions. Lack of drastic differentiation was attributable to the once existing connection between the Atlantic and the Amazonian forests at a non-distant past.

The primates of the Neotropics: genomes and chromosomes.

The classification of neotropical primates has been controversial. Different arrangements have been proposed, depending on taxonomic criteria and on the traits selected for phylogenetic reconstructions. These include gross morphologic characters, karyotypic attributes and DNA sequence data of nuclear and mitochondrial genes and of repetitive genomic components. These approaches have substantially clarified the main intergeneric relationships although several intrageneric arrangements still remain to be elucidated. In this review, we compare karyologic and molecular data of this speciose group.

Cytochrome b polymorphisms and population structure of two species of Alouatta (Primates).

We carried out a phylogenetic and population study in Alouatta caraya and Alouatta belzebul based on cytochrome b DNA sequence data. Maximum Parsimony and Median-Joining analyses grouped A. caraya from different localities showing a population structure in accordance with geographic distribution. The relation between A. caraya haplotypes could be explained with respect to the species range in the Cerrado, one of the most ancient morphoclimatic domains of South America, and the Chaco. Conversely, A. belzebul from the Amazonas and Atlantic forests grouped in a paraphyletic arrangement without an evident geographic pattern. Recent geologic events resulting in the separation of A. belzebul might explain why these geographically distant groups shared similar haplotypes and why ancestral polymorphisms might have been maintained in this species. Time of divergence estimates indicated that the splitting of the Alouatta lineage leading to A. caraya occurred some 4.58 MYA while the lineage leading to A. belzebul emerged 4.14 MYA.

Small mammals of Chapada dos Veadeiros National Park (Cerrado of Central Brazil): ecologic, karyologic, and taxonomic considerations.

This work is based on a survey of small mammals carried out in the Chapada dos Veadeiros National Park, a natural reserve located in the mountains of the Planalto Central Goiano in the Cerrado of Central Brazil. The 227 specimens collected represented six marsupial and 13 rodent species. Taxonomic, karyologic, and ecologic considerations are present and discussed in the present work. Our data reflected the faunal heterogeneity with respect to both elevation and vegetation because only eight of the 19 species were collected at both high and low elevations. The composition of the small mammal fauna of the park is influenced by predominance of forest formations at low elevations and cerrado with rupestrian areas at high elevations. Presence of endemic species and one undescribed demonstrated that the cerrado has an endemic fauna and a little known diversity of small mammals.

Molecular analysis of HPRT1(+) somatic cell hybrids derived from a carrier of an HPRT1 mutation responsible for Lesch-Nyhan syndrome.

Heterozygous carriers of HPRT1 mutations responsible for Lesch-Nyhan syndrome can be detected by analysis of somatic cell hybrids derived from peripheral blood lymphocytes and Hprt1-negative cells of rodent origin followed by selection in culture medium containing hypoxanthine, aminopterine, and thymidine (HAT). The parental origin of the X chromosome containing the normal HPRT1 allele in HPRT1(+) hybrid cell lines can be determined by molecular haplotyping attributable to highly polymorphic X-linked markers. We used this procedure to study a presumed carrier whose paternal active X chromosome always segregated in the cell hybrids derived from her. Conversely, her maternal X chromosome was systematically absent in most cell hybrids, or when present, it was inactive and coexisted with an active, paternal X chromosome. These results clearly demonstrated that the proband was a heterozygous carrier of a mutation responsible for HPRT1 deficiency.

Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by "exon by exon" PCR mediated SSCP analysis.

AIMS: To carry out a retrospective study, screening for mutations of the entire coding region of RB1 and adjacent intronic regions in patients with retinoblastoma. METHODS: Mutation screening in DNA extracts of formalin fixed, paraffin wax embedded tissues of 28 patients using combined "exon by exon" polymerase chain reaction mediated single strand conformational polymorphism analysis, followed by DNA sequencing. RESULTS: Eleven mutations were found in 10 patients. Ten mutations consisted of single base substitutions; 10 were localised in exonic regions (eight nonsense, one missense, and one frameshift) and another one in the intron-exon splicing region. Three novel mutations were identified: a 2 bp insertion in exon 2 (g.5506-5507insAG, R73fsX77), a G to A transition affecting the last invariant nucleotide of intron 13 (g.76429G>A), and a T to C transition in exon 20 (g.156795T>C, L688P). In addition, eight C to T transitions, resulting in stop codons, were found in five different CGA codons (g.64348C>T, g.76430C>T, g.78238C>T, g.78250C>T, and g.150037C>T). Although specific mutation hotspots have not been identified in the literature, eight of the 11 mutations occurred in CGA codons and seven fell within the E1A binding domains (codons 393-572 and 646-772), whereas five were of both types-in CGA codons within E1A binding domains. CONCLUSIONS: CGA codons and E1A binding domains are apparently more frequent mutational targets and should be initially screened in patients with retinoblastoma. Paraffin wax embedded samples proved to be valuable sources of DNA for retrospective studies, providing useful information for genetic counselling.

Small non-flying mammals from conserved and altered areas of Atlantic forest and Cerrado: comments on their potential use for monitoring environment.

Two Atlantic Forests and two Cerrado areas in Brazil were sampled for non-flying small mammal fauna. In each biome one area with altered and another with almost unaltered vegetation (national parks), were chosen to investigate these fauna. Species richness of Atlantic Forest and Cerrado was comparable in the conserved as well as in the altered areas. Data suggested that species could be divided into different ecological categories according to distribution, use of altered and/or relatively unaltered vegetation and habitat specificity. Within these ecological categories some species are appropriate indicators for monitoring environmental quality and degradation. Useful guidelines for wildlife management planning, including selecting areas for conservation units and their better boundary delimitation can ensue.

Genetic characterization of a Juquitiba-like viral lineage in Oligoryzomys nigripes in Rio de Janeiro, Brazil.

Hantaviruses, family Bunyaviridae, are rodent-borne RNA viruses that have caused cases of hantavirus cardiopulmonary syndrome (HCPS) in various regions of the Americas. There are five hantaviral lineages associated with HCPS in Brazil: Juquitiba virus (JUQV), Araraquara virus (ARAV), Laguna Negra-like virus (LNV), Castelo dos Sonhos virus (CASV), and Anajatuba virus (ANAJV). Three additional hantaviruses have been described in rodents alone: Rio Mearim virus, Jaborá virus, and a hantavirus lineage related to Seoul virus. This study describes the genetic detection and characterization of a Juquitiba-like hantavirus in Oligoryzomys nigripes, or the black-footed pygmy rice rat, in the Serra dos Orgãos National Park, Rio de Janeiro State, where so far no cases of HCPS have been reported.

Reproductive isolation due to the genetic incompatibilities between Thrichomys pachyurus and two subspecies of Thrichomys apereoides (Rodentia, Echimyidae).

We tested intrinsic reproductive isolation between 3 taxa of the South American caviomorph rodent Thrichomys (Rodentia, Echimyidae): T. pachyurus, T. apereoides subsp. apereoides and T. apereoides subsp. laurentius. They were mated in captivity and produced viable progeny. Some F1 hybrid females were fertile, whereas all F1 males were sterile. Histological examination revealed meiotic arrest at the primary spermatocyte stage. No sperm was detected in testes or epididymes. Electron microscopic analysis of surface spread synaptonemal complexes revealed a complete failure of chromosome pairing in F1 hybrids of T. pachyurus with T. apereoides subsp. laurentius and T. apereoides subsp. apereoides. In the male hybrids between T. apereoides subsp. apereoides and T. apereoides subsp. laurentius, meiosis did not proceed beyond diplotene, although all of the chromosomes, including heteromorphic ones, paired in an orderly fashion. Backcross males with homomorphic karyotypes showed segregation in meiosis progression. This indicates that male hybrid sterility is due to genetic, but not chromosomal, incompatibility of the parental taxa.

Molecular phylogeny of the genus Oryzomys (Rodentia: Sigmodontinae) based on cytochrome b DNA sequences.

The genus Oryzomys comprises 40 species arranged in several species groups. To test the monophyly of three Oryzomys species groups ("capito," nitidus, and subflavus), we analyzed, by distance, parsimony, and maximum-likelihood (ML), 801 bp of the mitochondrial gene cytochrome b. Our results did not sustain the monophyly of Oryzomys nor of the nitidus and subflavus species groups. Within the "capito" species group, O. perenensis appeared as a valid species, as a sister branch of the clade formed by O. megacephalus and O. laticeps. Within the nitidius species group, only the association between O. nitidus and O. lamia was well supported. The subflavus species group split into two clades: one with O. subflavus karyomorphotypes and another grouping O. angouya with species of different genera in the parsimony, distance, and ML trees.

Molecular phylogenetics of howler monkeys (Alouatta, Platyrrhini). A comparison with karyotypic data.

Molecular phylogenetic analyses of seven Brazilian Alouatta species, based on cytochrome b DNA sequence data were carried out. Parsimony and neighbor joining topologies grouped Alouatta belzebul and A. fusca as sister groups in one clade while another, well-supported clade contained A. seniculus as the most basal offshoot, followed by A. nigerrima as a sister lineage of A. macconnelli/A. stramineus. Estimates of inter-specific sequence divergence were generally low, and estimates of the time of divergence indicated that the main Alouatta lineages emerged during a short evolutionary interval. A comparison with karyotypic data confirmed the molecular topology showing a closer relationship between A. macconnelli and A. stramineus in respect to A. nigerrima. It also showed that the XX/XY sex chromosome system was maintained in several lineages while the X1X2Y/X1X1X2X2 system appeared independently at least three times during the radiation of howler monkeys. Moreover, the X1X2Y1Y2/X1X1X2X2 system might have appeared once or, alternatively, twice and independently.

Pericentric inversion in natural populations of Oligoryzomys nigripes (Rodentia: Sigmodontinae).

We analysed polymorphism for pericentric inversion in chromosome 3 of Oligoryzomys nigripes (Rodentia: Sigmodontinae) in several populations in Brazil and examined the meiotic behaviour of this chromosome in heterozygotes. We observed an orderly pairing of all chromosomes at pachytene in heterozygotes for the inverted chromosome 3. No indication of meiotic arrest and germ-cell death was found. Electron microscopy of synaptonemal complexes and conventional meiotic analysis indicated strictly nonhomologous synapsis and crossing-over suppression in the inverted region in the heterozygotes, which prevent the formation of unbalanced gametes. Thus, the pericentric inversion in chromosome 3 does not apparently result in any selective disadvantages in heterozygous carriers. In the majority of the populations studied, the frequencies of acrocentric homozygotes, metacentric homozygotes, and heterozygotes were in Hardy-Weinberg equilibrium. However, in some populations, we detected an excess of heterozygotes and a deficiency of acrocentric homozygotes.

Chromosome painting in Callicebus lugens, the species with the lowest diploid number (2n=16) known in primates.

Cytogenetic studies have shown that New World primates are karyologically diverse and highly derived. The genus Callicebus is the best example of this karyological diversity, with diploid numbers ranging from 2n=50 to 2n=16. We report on Callicebus lugens, which has the lowest diploid number (2n=16) yet found in the primate order and represents a striking example of extreme karyotypic shuffling. To better understand the genomic rearrangements that have resulted in this extremely low diploid number, we mapped chromosome homologies between C. lugens and humans by in situ hybridization. The total number of hybridization signals was 42, excluding the Y chromosome, with a total of 34 syntenic associations not found in humans. This species has one of the most derived karyotypes among the Platyrrhini. Fusion has been the predominant mode of karyological evolution, although fissions and inversions have also transformed the C. lugens karyotype. Remarkably in such a highly rearranged karyotype, the synteny of 11 human chromosomes (4, 5, 9, 12, 13, 14, 17, 18, 20, 21, and X) was maintained intact, even if most of these human-homologous gene clusters were translocated. Other human syntenies, such as homologues to human chromosomes 10 and 16, were highly fragmented. Comparisons of the C. lugens-human homology map with those of other New World primates have not yet helped establish a phylogenic arrangement between congeneric species or link Callicebus with any other genus.

Induction and characterization of hypoxanthine-phosphoribosyltransferase (Hprt-) deficient cell lines of Akodon cursor (Rodentia, Sigmodontinae).

Hypoxanthine-phosphoribosyltransferase negative (Hprt-) cell lines derived from an Akodon cursor liposarcoma were obtained by induced mutagenesis. All but one Hprt- cell line lacked Hprt mRNA transcripts while one (AKO 3) coded for a truncated protein. Cell fusion and karyotypic analyses showed that one cell line (AKO 1-15) could be successfully used for constructing hybrid panels and allow for a clear identification of human chromosomes in hybrid cells.

The chromosomes of Nectomys (Rodentia, Cricetidae) with 2n = 52, 2n = 56, and interspecific hybrids (2n = 54)

Karyologic comparisons of Nectomys with 2n = 52 and 2n = 56, captured in different regions of Brazil, revealed the existence of two presumptive tandem fusions. These differences proved to represent drastic reproductive barriers between these two karyologic groups, based on an analysis of spermatogenesis in captive-bred hybrids (2n = 54). In these hybrid specimens, cytologic and histologic analyses indicated severe meiotic arrest and the presence of two trivalents in the first meiotic division, in which 26 elements were frequently observed. These findings and the sterility of crosses between F1 males and females as well as backcrosses indicated that Nectomys populations with 2n = 52 and 2n = 56 should be considered different species and that the present taxonomic arrangement of this genus must be reviewed.

Karyologic evidence of diversification of the genus Thrichomys (Rodentia, Echimyidae).

Karyologic analysis of Thrichomys specimens from different Brazilian localities, in Pantanal, Cerrado and Caatinga biomes, shows different chromosome complements. The 2n = 34, FN = 64 karyotype is found in Mato Grosso do Sul state; the 2n = 30, FN = 54 karyotype in Bahia, Pernambuco, Piauí and Ceará states; the 2n = 30, FN = 56 karyotype in Goiás and Tocantins states, the 2n = 28, FN = 50 karyotype in Minas Gerais state, the 2n = 28, FN = 52 and 2n = 26, FN = 48 karyotypes in Bahia state. Comparisons of G-band patterns allowed the identification of homologies shared by all karyotypes and show that the two karyotypes with the lowest diploid number (2n = 26 and 2n = 28) belong to two different evolutionary lineages. The most proper names for each karyomorphic population are: Thrichomys pachyurus for 2n = 34; Thrichomys apereoides apereoides for 2n = 28, FN = 50; Thrichomys apereoides laurentius for 2n = 30, FN = 54 and Thrichomys inermis for 2n = 26. Two karyotypes (2n = 28, FN = 52 and 2n = 30, FN = 56) could not be attributed to any subspecies. These different karyomorphotypes are allopatric and/or parapatric.

Molecular and karyologic variation in the genus Isothrix (Rodentia, Echimyidae).

Phylogenetic relationships among available samples of the genus Isothrix using mitocondrial gene cytochrome b were carried out and a new karyotype of Isothrix specimens referred to Isothrix negrensis from the mid course of Rio Negro, in the Brazilian Amazon, was described. Molecular, karyological and morphological data of Isothrix negrensis was compared to that of I. bistriata and I. pagurus. Cytochrome b DNA sequence analyses clearly separate I. negrensis from I. bistriata and genetic distances estimates between then are greater than between species of the related genus Mesomys. The median-joining analysis postulated the presence of two median vectors between I. bistriata haplotypes from adjacent localities, suggesting that genetic isolation between them is unrelated to geographic distance. These results confirm previous molecular differences suggesting that I.bistriata is a composite of several taxa. The karyotype of Isothrix negrensis also differs from those reported for I. bistriata and I. pagurus. In relation to the external morphology I. negrensis differs from I. bistriata by the overall darker dorsal coloration, head darker than dorsum, presence of a longitudinal line of blackish hairs in the proximal part of the tail extending until near to its base, dorsal surface of hind feet covered with yellowish hairs and stripe of light hairs in the head reduced to a small patch.

Gene assignment in the spider monkey (Ateles paniscus chamek--APC): APE-MYH7 to 2q; AR-GLA-F8C to the X chromosome.

Comparative gene assignment between the spider monkey species Ateles paniscus chamek (APC) and man (HSA) showed conserved syntenic associations despite extensive karyotypic rearrangement between species. Two HSA 14q genes were allocated to APC 2q, being syntenic to other HSA 14q and HSA 15q markers previously assigned to APC 2q, and to HSA 12q genes previously assigned to APC 2p. These findings were consistent with A. geoffroyi chromosome painting with human whole-chromosome probes, indicating that the genus Ateles is karyotypically very rearranged. On the other hand, three human X-linked markers were assigned to the Ateles X chromosome, indicating that this chromosome is evolutionary stable.