Identification of Novel Cellular Transcription Factors that Regulate Early Promoters of Human Papillomavirus Types 18 and 16

Background. The long control region (LCR) of human papillomavirus (HPV) regulates early gene transcription by interaction with several viral and cellular transcription factors (TFs). Methods. To identify novel TFs that could influence early expression of HPV type 18 (HPV-18) and HPV type 16 (HPV-16), a high-throughput transfection array was used. Results. Among the 704 TFs tested, 28 activated and 36 inhibited the LCR of HPV-18 by more than 2-fold. For validation, C33 cells were cotransfected with increasing amounts of selected TF expression plasmids in addition to LCR-luciferase vectors of different molecular variants of HPV-18 and HPV-16. Among the TFs identified, only GATA3, FOXA1, and MYC have putative binding sites within the LCR sequence, as indicated using the TRANSFAC database. Furthermore, we demonstrated FOXA1 and MYC in vivo binding to the LCR of both HPV types using chromatin immunoprecipitation assay. Conclusions. We identified new TFs implicated in the regulation of the LCR of HPV-18 and HPV-16. Many of these factors are mutated in cancer or are putative cancer biomarkers and could potentially be involved in the regulation of HPV early gene expression.

Weedy Hosts and Prevalence of Potential Leafhopper Vectors (Hemiptera: Cicadellidae) of a Phytoplasma (16SrIX group) Associated With Huanglongbing Symptoms in Citrus Groves

Huanglongbing (HLB) is a severe citrus (Citrus spp.) disease associated with the bacteria genus Candidatus Liberibacter, detected in Brazil in 2004. Another bacterium was found in association with HLB symptoms and characterized as a phytoplasma belonging to the 16SrIX group. The objectives of this study were to identify potential leafhopper vectors of the HLB-associated phytoplasma and their host plants. Leafhoppers were sampled every other week for 12 mo with sticky yellow cards placed at two heights (0.3 and 1.5 m) in the citrus tree canopy and by using a sweep net in the ground vegetation of two sweet orange, Citrus sinensis (L.) Osbeck, groves infected by the HLB-phytoplasma in Sao Paulo state. Faunistic analyses indicated one Agalliinae (Agallia albidula Uhler) and three Deltocephalinae [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (Stal), and Scaphytopius (Convelinus) marginelineatus (Stal)] species, as the most abundant and frequent leafhoppers (Hemiptera: Cicadellidae). Visual observations indicated an association of leafhopper species with some weeds and the influence of weed species composition on leafhopper abundance in low-lying vegetation. S. marginelineatus and P. flavicosta were more frequent on Sida rhombifolia L. and Althernantera tenella Colla, respectively, whereas A. albidula was observed more often on Conyza bonariensis (L.) Cronq. and B. hebe only occurred on grasses. DNA samples of field-collected S. marginelineatus were positive by polymerase chain reaction and sequencing tests for the presence of the HLB-phytoplasma group, indicating it as a potential vector. The association of leafhoppers with their hosts may be used in deciding which management strategies to adopt against weeds and diseases in citrus orchards.

Spatial distribution of arboviral mosquito vectors (Diptera, Culicidae) in Vale do Ribeira in the South-eastern Brazilian Atlantic Forest

Mosquitoes are vectors of arboviruses that can cause encephalitis and hemorrhagic fevers in humans. Aedes serratus (Theobald), Aedes scapularis (Rondani) and Psorophora ferox (Von Humboldt) are potential vectors of arboviruses and are abundant in Vale do Ribeira, located in the Atlantic Forest in the southeast of the State of Sao Paulo, Brazil. The objective of this study was to predict the spatial distribution of these mosquitoes and estimate the risk of human exposure to mosquito bites. Results of the analyses show that humans are highly exposed to bites in the municipalities of Cananeia, Iguape and Ilha Comprida. In these localities the incidence of Rocio encephalitis was 2% in the 1970s. Furthermore, Ae. serratus, a recently implicated vector of yellow fever virus in the State of Rio Grande do Sul, should be a target for the entomological surveillance in the southeastern Atlantic Forest. Considering the continental dimensions of Brazil and the inherent difficulties in sampling its vast area, the habitat suitability method used in the study can be an important tool for predicting the distribution of vectors of pathogens.

MICROSATELLITE MARKERS DEVELOPED FOR UTRICULARIA RENIFORMIS (LENTIBULARIACEAE)

Premise of the study: Microsatellite markers were developed to study the genetic diversity and population structure of the carnivorous bladderwort Utricularia reniformis, which is endemic to the Atlantic Forest of southern and southeastern Brazil. Cross-species amplification was tested in U. gibba, U. neottioides, U. subulata, and Pinguicula benedicta. Methods and Results: The U. reniformis genome was sequenced in a 454 GS FLX sequencer, and eight primer sets were developed based on the microsatellites identified from the reads. All loci are polymorphic, showing 1.6 to 4.8 alleles per population. Preliminary results show that primer sets are suitable for population-level studies. Cross-species amplifi cation was successful in three other Utricularia species and one Pinguicula species. Conclusions: Markers developed in this study provide tools for analyses of intra- and interpopulation genetic diversity in Utricularia and Pinguicula.

Morphometrical diagnosis of the malaria vectors Anopheles cruzii, An. homunculus and An. bellator

Background: Anopheles (Kerteszia) cruzii is a primary vector of Plasmodium parasites in Brazil's Atlantic Forest. Adult females of An. cruzii and An. homunculus, which is a secondary malaria vector, are morphologically similar and difficult to distinguish when using external morphological characteristics only. These two species may occur syntopically with An. bellator, which is also a potential vector of Plasmodium species and is morphologically similar to An. cruzii and An. homunculus. Identification of these species based on female specimens is often jeopardised by polymorphisms, overlapping morphological characteristics and damage caused to specimens during collection. Wing geometric morphometrics has been used to distinguish several insect species; however, this economical and powerful tool has not been applied to Kerteszia species. Our objective was to assess wing geometry to distinguish An. cruzii, An. homunculus and An. bellator. Methods: Specimens were collected in an area in the Serra do Mar hotspot biodiversity corridor of the Atlantic Forest biome (Cananeia municipality, State of Sao Paulo, Brazil). The right wings of females of An. cruzii (n= 40), An. homunculus (n= 50) and An. bellator (n= 27) were photographed. For each individual, 18 wing landmarks were subjected to standard geometric morphometrics. Discriminant analysis of Procrustean coordinates was performed to quantify wing shape variation. Results: Individuals clustered into three distinct groups according to species with a slight overlap between representatives of An. cruzii and An. homunculus. The Mahalanobis distance between An. cruzii and An. homunculus was consistently lower (3.50) than that between An. cruzii and An. bellator (4.58) or An. homunculus and An. bellator (4.32). Pairwise cross-validated reclassification showed that geometric morphometrics is an effective analytical method to distinguish between An. bellator, An. cruzii and An. homunculus with a reliability rate varying between 78-88%. Shape analysis revealed that the wings of An. homunculus are narrower than those of An. cruzii and that An. bellator is different from both of the congeneric species. Conclusion: It is possible to distinguish among the vectors An. cruzii, An. homunculus and An. bellator based on female wing characteristics.

Geographical distribution of American cutaneous leishmaniasis and its phlebotomine vectors (Diptera: Psychodidae) in the state of São Paulo, Brazil

Abstract Background American cutaneous leishmaniasis (ACL) is a re-emerging disease in the state of São Paulo, Brazil. It is important to understand both the vector and disease distribution to help design control strategies. As an initial step in applying geographic information systems (GIS) and remote sensing (RS) tools to map disease-risk, the objectives of the present work were to: (i) produce a single database of species distributions of the sand fly vectors in the state of São Paulo, (ii) create combined distributional maps of both the incidence of ACL and its sand fly vectors, and (iii) thereby provide individual municipalities with a source of reference material for work carried out in their area. Results A database containing 910 individual records of sand fly occurrence in the state of São Paulo, from 37 different sources, was compiled. These records date from between 1943 to 2009, and describe the presence of at least one of the six incriminated or suspected sand fly vector species in 183/645 (28.4%) municipalities. For the remaining 462 (71.6%) municipalities, we were unable to locate records of any of the six incriminated or suspected sand fly vector species (Nyssomyia intermedia, N. neivai, N. whitmani, Pintomyia fischeri, P. pessoai and Migonemyia migonei). The distribution of each of the six incriminated or suspected vector species of ACL in the state of São Paulo were individually mapped and overlaid on the incidence of ACL for the period 1993 to 1995 and 1998 to 2007. Overall, the maps reveal that the six sand fly vector species analyzed have unique and heterogeneous, although often overlapping, distributions. Several sand fly species - Nyssomyia intermedia and N. neivai - are highly localized, while the other sand fly species - N. whitmani, M. migonei, P. fischeri and P. pessoai - are much more broadly distributed. ACL has been reported in 160/183 (87.4%) of the municipalities with records for at least one of the six incriminated or suspected sand fly vector species, while there are no records of any of these sand fly species in 318/478 (66.5%) municipalities with ACL. Conclusions The maps produced in this work provide basic data on the distribution of the six incriminated or suspected sand fly vectors of ACL in the state of São Paulo, and highlight the complex and geographically heterogeneous pattern of ACL transmission in the region. Further studies are required to clarify the role of each of the six suspected sand fly vector species in different regions of the state of São Paulo, especially in the majority of municipalities where ACL is present but sand fly vectors have not yet been identified.