Zmienność genetyczna, specyficzność żywicielska i zdolność do przenoszenia wirusa mozaiki smugowatej pszenicy (WSMV) w obrębie kompleksu gatunków Aceria tosichella

Wydział Biologii: Instytut Biologii Środowiska

Caracterização agronômica e fisíco-química de progênies de maracujazeiro azedo (Passiflora edulis Sims) no Distrito Federal

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Agronomia, 2016.

Sinal amarelo.

2016

Potyvirus causando mosaico em plantas daninhas nas culturas do milho e do sorgo.

2016

Gwas analysis for the identification of molecular basis responsible for yield related traits and disease resistance in durum wheat

The PhD thesis was developed in the framework of Innovar H2020 project. This project aimed at using genomics, transcriptomics and phenotyping techniques to update varietal registration procedure used in Europe for Value of Cultivation and Use (VCU) and Distinctiness Uniformity and Stability (DUS) protocols. The phenotypic and genotypic diversity of a durum wheat panel were assessed for different agronomic traits, connected with wheat development, disease resistance and spike fertility. A panel of 253 durum wheat varieties was characterized for VCU and DUS traits and genotyped with Illumina 90K SNP Chip array (Wang et al., 2014). GWAS analysis was performed, detecting strong QTLs confirmed also by literature review. Candidate genes were identified for each trait and molecular markers will be developed to be used for marker assisted selection in breeding programs. As for disease resistance, the panel was evaluated for resistance to Soil-Borne-Cereal-Mosaic-Virus (SBCMV). A major QTL, sbm2, was detected on chromosome 2B responsible for durum wheat resistance (Maccaferri et al., 2011). The sbm2 interval was explored by fine mapping on segregant population using KASP markers and by RNASeq analysis, detecting candidate genes involved in plant-pathogen reaction. As regards yield related traits, detailed analysis was performed on the GNI-2A QTL (Milner et al., 2016), responsible for increased number spike fertility. Fine mapping analysis was performed on durum panel identifying hox2 a strong candidate gene, codifying for transcription factor protein. The gene is paralogue of GNI-1 (Sakuma et al., 2019), and it has a 4 kbp deletion responsible for increased number of florets per spikelet. To conclude, the herein reported thesis shows a complete characterization of agronomic and disease resistance traits in modern durum wheat varieties. The results obtained will augment available information for each variety, identifying informative molecular markers for breeding purposes and QTLs/candidate genes responsible for different agronomic traits.

Natural infection of Nicandra physaloides by Tomato severe rugose virus in Brazil

Nicandra physaloides, a common weed in South America, was found to be infected by an isolate of Tomato severe rugose virus (ToSRV), a bipartite begomovirus. The plants developed severe yellow rugose mosaic and were collected in So Paulo State, Brazil. This isolate of ToSRV was transmitted by Bemisia tabaci B biotype from infected plants of N. physaloides to healthy plants of N. physaloides and tomato in a glasshouse. This is the first report of natural infection of N. physaloides by ToSRV in Brazil.

Lymphocyte subset analyses in healthy adults vaccinated with yellow fever 17DD virus

In this study the kinetics of humoral and cellular immune responses in first-time vaccinees and re-vaccinees with the yellow fever 17DD vaccine virus was analyzed. Flow cytometric analyses were used to determine percentual values of T and B cells in parallel to the yellow fever neutralizing antibody production. All lymphocyte subsets analyzed were augmented around the 30th post vaccination day, both for first-time vaccinees and re-vaccinees. CD3+ T cells increased from 30.8% (SE ± 4%) to 61.15% (SE ± 4.2%), CD4+ T cells from 22.4% (SE ± 3.6%) to 39.17% (SE ± 2%) with 43% of these cells corresponding to CD4+CD45RO+ T cells, CD8+ T cells from 15.2% (SE ± 2.9%) to 27% (SE ± 3%) with 70% corresponding to CD8+CD45RO+ T cells in first-time vaccinees. In re-vaccinees, the CD3+ T cells increased from 50.7% (SE ± 3%) to 80% (SE ± 2.3%), CD4+ T cells from 24.9% (SE ± 1.4%) to 40% (SE ± 3%) presenting a percentage of 95% CD4+CD45RO+ T cells, CD8+ T cells from 19.7% (SE ± 1.8%) to 25% (SE ± 2%). Among CD8+CD38+ T cells there could be observed an increase from 15 to 41.6% in first-time vaccinees and 20.7 to 62.6% in re-vaccinees. Regarding neutralizing antibodies, the re-vaccinees presented high titers even before re-vaccination. The levels of neutralizing antibodies of first-time vaccinees were similar to those presented by re-vaccinees at day 30 after vaccination, indicating the success of primary vaccination. Our data provide a basis for further studies on immunological behavior of the YF 17DD vaccine.

Retention of a recombinant GFP protein expressed by the yellow fever 17D virus in the E/NS1 intergenic region in the endoplasmic reticulum

The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein.

Circulation of a Meaban-like virus in yellow-legged gulls and seabird ticks in the western Mediterranean Basin

In recent years, a number of zoonotic flaviviruses have emerged worldwide, and wild birds serve as their major reservoirs. Epidemiological surveys of bird populations at various geographical scales can clarify key aspects of the eco-epidemiology of these viruses. In this study, we aimed at exploring the presence of flaviviruses in the western Mediterranean by sampling breeding populations of the yellow-legged gull (Larus michahellis), a widely distributed, anthropophilic, and abundant seabird species. For 3 years, we sampled eggs from 19 breeding colonies in Spain, France, Algeria, and Tunisia. First, ELISAs were used to determine if the eggs contained antibodies against flaviviruses. Second, neutralization assays were used to identify the specific flaviviruses present. Finally, for colonies in which ELISA-positive eggs had been found, chick serum samples and potential vectors, culicid mosquitoes and soft ticks (Ornithodoros maritimus), were collected and analyzed using serology and PCR, respectively. The prevalence of flavivirus-specific antibodies in eggs was highly spatially heterogeneous. In northeastern Spain, on the Medes Islands and in the nearby village of L'Escala, 56% of eggs had antibodies against the flavivirus envelope protein, but were negative for neutralizing antibodies against three common flaviviruses: West Nile, Usutu, and tick-borne encephalitis viruses. Furthermore, little evidence of past flavivirus exposure was obtained for the other colonies. A subset of the Ornithodoros ticks from Medes screened for flaviviral RNA tested positive for a virus whose NS5 gene was 95% similar to that of Meaban virus, a flavivirus previously isolated from ticks of Larus argentatus in western France. All ELISA-positive samples subsequently tested positive for Meaban virus neutralizing antibodies. This study shows that gulls in the western Mediterranean Basin are exposed to a tick-borne Meaban-like virus, which underscores the need of exploring the spatial and temporal distribution of this flavivirus as well as its potential pathogenicity for animals and humans.

Dinâmica temporal e espacial da begomovirose causada por Tomato yellow vein streak virus em tomateiro na região de Campinas-SP

O objetivo desse trabalho foi caracterizar os padrões temporal e espacial do Tomato yellow vein streak virus (ToYVSV) em tomatais cultivados em condições de campo, no município de Sumaré, e de estufa plástica, na região de Elias Fausto, Estado de São Paulo. No ensaio de campo, plantado com a variedade Alambra, foram avaliadas 4.032 plantas, distribuídas em oito blocos. Em oito estufas plásticas, com plantios escalonados da variedade Ikram, foram avaliadas 6.016 plantas. As avaliações foram feitas com base nos sintomas característicos induzidos por esse vírus. A confirmação da identidade do vírus foi feita por meio da análise da seqüência de nucleotídeos de parte do DNA-A viral (genes AV1 e AC3). No ensaio em condições de campo, a incidência da doença evoluiu lentamente, desde um mínimo de 0,002 (proporção de plantas sintomáticas) até um máximo de 0,0497. Mesmo assim, foi possível constatar um efeito de borda, pois a incidência média de plantas doentes nos blocos situados nos bordos da área foi 2,1 vezes maior do que naqueles internos. O progresso da incidência da doença foi linear, o que indica que novas infecções foram devidas principalmente a um influxo constante de vetores virulíferos de fora para dentro da área avaliada. Nos plantios em estufas plásticas, os níveis finais de doença foram fortemente dependentes da época de plantio, com médias variando de 4,8% a 69,3%. A distribuição espacial de plantas sintomáticas nesses plantios foi fortemente agregada. Essa agregação provavelmente não se deve a infecções secundárias dentro das estufas plásticas, mas sim à concentração de plantas sintomáticas nos bordos das estufas, conseqüência da migração de vetores virulíferos a partir de áreas externas à estufa. Com base nesses resultados, sugere-se a eliminação de fontes de inóculo representadas por plantios mais velhos de tomateiro e por hospedeiras do vírus na vegetação espontânea como uma das principais medidas para o manejo da doença.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Mosquito (Diptera: Culicidae) Diversity of a Forest-Fragment Mosaic in the Amazon Rain Forest

To study the impact of Amazonian forest fragmentation on the mosquito fauna, an inventory of Culicidae was conducted in the upland forest research areas of the Biological Dynamics of Forest Fragments Project located 60 km north of Manaus, Amazonas, Brazil. The culicid community was sampled monthly between February 2002 and May 2003. CDC light traps, flight interception traps, manual aspiration, and net sweeping were used to capture adult specimens along the edges and within forest fragments of different sizes (1, 10, and 100 ha), in second-growth areas surrounding the fragments and around camps. We collected 5,204 specimens, distributed in 18 genera and 160 species level taxa. A list of mosquito taxa is presented with 145 species found in the survey, including seven new records for Brazil, 16 new records for the state of Amazonas, along with the 15 morphotypes that probably represent undescribed species. No exotic species [Aedes aegypti (L.) and Aedes albopictus (Skuse)] were found within the sampled areas. Several species collected are potential vectors of Plasmodium causing human malaria and of various arboviruses. The epidemiological and ecological implications of mosquito species found are discussed, and the results are compared with other mosquito inventories from the Amazon region.

Rabies virus in a pregnant naturally infected southern yellow bat (Lasiurus ega)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.