RESUMEN
A novel virus was discovered in a freeze-dried collection held at SASA, UK, originating from potato (Solanum tuberosum) cv. Nadine. The complete sequence of the viral RNA was determined to be 3674 nucleotides in length encoding five predicted proteins. Based on the deduced genome organization and phylogenetic analysis, this virus represents a putative new member of the genus Alphanecrovirus, family Tombusviridae, most closely related to isolates of Olive mild mosaic virus. The virus was easily transmitted to indicator plants with symptoms that were slower to develop and less severe than those of related viruses. To distinguish this virus, the clearest symptom differences occurred with Nicotiana debneyi, Chenopodium amaranticolor and Ch. quinoa. The virus was detected with antisera to the related viruses tobacco necrosis virus A and tobacco necrosis virus D. The close association to the tobacco necrosis viruses would suggest this virus is not a new introduction to potato but in the past has been misidentified as one of these viruses. The virus isolate has been named potato necrosis virus.
Asunto(s)
Genoma Viral , Filogenia , ARN Viral/genética , Solanum tuberosum/virología , Tombusviridae/genética , Chenopodium/virología , Chenopodium quinoa/virología , Efecto Fundador , Sistemas de Lectura Abierta , Enfermedades de las Plantas/virología , Nicotiana/virología , Tombusviridae/clasificación , Tombusviridae/aislamiento & purificación , Tombusviridae/patogenicidad , Reino UnidoRESUMEN
Viroids can cause diseases of considerable economic importance; in Europe the main concern is with pospiviroids that may affect the tomato and potato industries. Methods for detection are required that are both sensitive and robust. The detection method described here is a probe hybridization method with a commercially available digoxigenin (DIG) labelled full-length Potato spindle tuber viroid (PSTVd) RNA probe. This method detects PSTVd and all other known pospiviroids.
Asunto(s)
Digoxigenina/química , Enfermedades de las Plantas/microbiología , Tubérculos de la Planta/virología , Sondas ARN/química , ARN Viral/análisis , Solanum tuberosum/virología , Viroides/aislamiento & purificación , ARN Viral/genética , Especificidad de la Especie , Viroides/clasificación , Viroides/genéticaRESUMEN
To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full-length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The alphaSIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the alpha-CH3 domain from human IgA. To express the full-length IgA, the individual light and heavy chains from the TGEV-specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co-infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant-expressed alphaSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody-expressing plant tissue to 2-day-old piglets showed that both the alphaSIP and full-length IgA molecules can provide in vivo protection against TGEV.
Asunto(s)
Anticuerpos/inmunología , Comovirus/genética , Coronavirus/inmunología , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismo , Proteínas de Plantas/inmunología , Potexvirus/genética , Animales , Anticuerpos/genética , Anticuerpos/metabolismo , Vectores Genéticos/genética , Inmunoglobulina A/genética , Región Variable de Inmunoglobulina/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Porcinos , Transfección/métodosRESUMEN
To investigate the potential of antibody derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed a small immune protein (SIP) in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The epsilonSIP molecule consisted of a single-chain antibody (scFv) specific for the porcine coronavirus transmissible gastroenteritis virus (TGEV) linked to the epsilon-CH4 domain from human immunoglobulin E (IgE). In some constructs, the sequence encoding the epsilonSIP molecule was flanked by the leader peptide from the original murine antibody at its N-terminus and an endoplasmic reticulum retention signal (HDEL) at its C-terminus to allow the expressed protein to be directed to, and retained within, the endoplasmic reticulum. Western blot analysis of samples from Nicotiana clevelandii or cowpea tissue infected with constructs revealed the presence of SIP molecules which retained their ability to dimerize. The analysis of crude plant extracts revealed that the plant-expressed epsilonSIP molecules could bind to and neutralize TGEV in tissue culture, the levels of binding and neutralization reflecting the level of expression. Oral administration of crude extracts from SIP-expressing plant tissue to 2-day-old piglets demonstrated that the extracts which showed the highest levels of in vitro neutralization could also provide in vivo protection against challenge with TGEV.