Eliana Borges Rivas

Molecular characterization of Brazilian infectious bursal disease virus isolated from 1997 to 2005.

Abstract This retrospective study concerned 41 infectious bursal disease virus (IBDV) isolates obtained from Brazilian broiler and layers flocks by reverse transcription-polymerase chain reaction. Twenty-five of them were identified as very virulent (vv) by restriction enzyme analysis and by further nucleotide and phylogenetic analysis. All of them had the typical amino acid residues, and all clustered in a phylogenetic tree with the vvIBDV strains. Four amino acid substitutions, at positions D213N, G254D, S317R, and D323E, were common to 3 vv isolates, Br/03/DB, Br/03/CK, and Br/04/CR, and differed from other vv isolates and strains. These isolates came from the same locale, but were collected in different years, indicating that the vvIBDVs circulating on Brazilian farms are undergoing slight but continuous exchanges.

A new Badnavirus species detected in Bougainvillea in Brazil

Badnavirus in Bougainvillea spectabilis showing virus-like symptoms was identified by the presence of bacilliform particles, measuring 125–130 × 30–40 nm in leaf-dip preparations and by analysis of its putative open reading frame 3 sequence. The virus, tentatively named Bougainvillea bacilliform virus (BBV), had the highest identities (up to 60%) with Spiraea yellow leaf spot virus, Gooseberry vein banding associated virus, Taro bacilliform virus, and Citrus yellow mosaic virus. In phylogenetic analysis, BBV clustered with Badnavirus putative species. Attempts to transmit the virus to several hosts failed. This is the first report of a new Badnavirus detected in Bougainvillea.

Phylogenetic analysis of Tomato mosaic virus from Hemerocallis sp. and Impatiens hawkeri

The culture and commercialization of ornamental plants have considerably increased in the last years. To supply the commercial demand, several Hemerocallis and Impatiens varieties have been bred for appreciated qualities such as flowers with a diversity of shapes and colors. With the aim of characterizing the tobamovirus isolated from Hemerocallis sp. (tobamo-H) and Impatiens hawkeri (tobamo-I) from the USA and Sao Paulo, respectively, as well as to establish phylogenetic relationships between them and other Tobamovirus species, the viruses were submitted to RNA extraction, RT-PCR amplification, coat-protein gene sequencing and phylogenetic analyses. Comparison of tobamovirus homologous sequences yielded values superior to 98.5% of identity with Tomato mosaic virus (ToMV) isolates at the nucleotide level. In relation to tobamo-H, 100% of identity with ToMV from tomatoes from Australia and Peru was found. Based on maximum likelihood (ML) analysis it was suggested that tobamo-H and tobamo-I share a common ancestor with ToMV, Tobacco mosaic virus, Odontoglossum ringspot virus and Pepper mild mottle virus. The tree topology reconstructed under ML methodology shows a monophyletic group, supported by 100% of bootstrap, consisting of various ToMV isolates from different hosts, including some ornamentals, from different geographical locations. The results indicate that Hemerocallis sp. and I. hawkeri are infected by ToMV. This is the first report of the occurrence of this virus in ornamental species in Brazil.

Diversidad genética de aislamientos de Bacillus subtilis con potencial para el control biológico de Colletotrichum acutatum y Guignardia citricarpa

El sector citricola enfrenta serios problemas representados por enfermedades en las flores y en frutos jovenes que ademas de disminuir la productividad, devaluan los frutos por el aspecto que le dan a los mismos. Tales enfermedades estan representadas, principalmente, por la mancha negra de los frutos citricos (MNC) y por la caida prematura de los frutos citricos (CPFC), donde la medida predominante de control es la pulverizacion con productos quimicos. Entretanto, los costos financieros y ambientales de las aplicaciones con estes productos quimicos, sumado a las crecientes restricciones de la presencia de residuos, estan a exigir el estudio de nuevas alternativas de control. Entre estas, el control biologico surge como una alternativa importante. Estudios fueron anteriormente realizados, bajo condiciones de laboratorio y de campo, con el objetivo de determinar la potencialidad de aislados de Bacillus subtilis en el control de las enfermedades mencionadas arriba, puesto que, uno aislado, el ACB-69 fue el que presento la mejor eficiencia de control. Frente a lo anteriormente expuesto y, sabiendo que, el conocimiento de la biodiversidad de los seres es importante para la determinacion de sus funciones potenciales, el presente proyecto de investigacion tuvo como objetivo estudiar la diversidad genetica, a traves de marcadores moleculares AFLP, de 32 aislamientos de B. subtilis con la finalidad de se encontrar, dentro los mismos, uno (o mas aislados) que presentase mayor semejanza con el ACB-69 y que cuando testado, bajo condiciones naturales de ocurrencia de las enfermedades, si puede encontrar similar control. En funcion de los resultados experimentales obtenidos, se concluyo que: (a) los aislados de B. subtilis estudiados se agruparon en el filograma de distancia genetica, independiente de la procedencia o del huesped; (b) los aislados ACB-69 y ACB-83, con potencial para el control de la caida prematura de los frutos citricos, comparten la misma ancestralidad, lo que puede ser evaluado por la metodologia aplicada; c) en terminos biologicos, el aislado ACB-83 merece mas estudios cuanto a la viabilidad de control de las enfermedades caida prematura de los frutos citricos y de la mancha negra, bajo condiciones de campo.

A new Potyvirus species in Cotyledon orbiculata in mixed infection with a Nucleorhabdovirus.

Several species and varieties of the family Crassulaceae have been introduced and cultivated for ornamental as well as medicinal purposes in Brazil, more prominently Cotyledon orbiculata (pig’s ear). This work reports the identification and characterization of viruses associated with foliar mosaic and distortion of C. orbiculata from the succulent plant collection of the garden of the University of Sao Paulo, Brazil. In situ analyses revealed enveloped bacilliform particles in the perinuclear space of infected C. orbiculata cells, and cytoplasmic cylindrical inclusions, typical of the family Potyviridae. Viruses were mechanically transmitted and separated by differential indicator hosts and aphid transmission (Myzus persicae). Chenopodium amaranticolor and Datura stramonium served as differential hosts for the potyvirus and the bacilliform virus, respectively. Positive serological reactions were obtained when leaf extracts from naturally infected C. orbiculata and experimental hosts were exposed to an antiserum to Sonchus yellow net virus (genus Nucleorhabdovirus). Identification of the potyvirus by RT-PCR, cloning into pGEM-T vector and sequencing generated a 1,752 nt fragment corresponding to part of the nuclear inclusion protein b (NIb), the complete coat protein (CP) and the untranslated region (UTR). Amino acid identities below 80% were observed, indicating that the potyvirus of C. orbiculata may be a new species for which the name Cotyledon virus Y (CotVY) is suggested.

Partial VP1 sequencing of Brazilian infectious bursal disease virus strains

Infectious bursal disease virus (IBDV) is classified according to the antigenicity and virulence into classical virulent (cv), very virulent (vv), and antigenic variant strains. The molecular basis for the IBDV antigenic variation is well established and is associated to the capsid protein, VP2 (gene VP2 of segment A), whereas both VP2 and the RNA-dependent RNA polymerase, VP1 (gene VP1 of segment B), have been correlated with the virulence. In this study, seventeen Brazilian IBDV samples previously characterized by the VP2 gene as cv (three) and vv (fourteen) strains were genetically and molecularly analyzed for their VP1 gene. All of the strains kept with the same cv or vv classification except one sample, Br/03/DR. This sample was classified as vv by its VP2 gene, but it was most closely related to the cv strains by its VP1 partial sequence and phylogeny. Studies on the phylogeny of VP1 have suggested a possible reassortment event that originated the vvVP1. In this case, the sample carrying vvVP2 and cvVP1 could be a descendant of IBDV ancestors prior to the reassortment of vvVP1; alternatively, it could be the result of a genetic exchange between the segments of different strains or with a live attenuated vaccine. Nevertheless, this is the first report of natural genetic reassortment of IBDV in Brazil.