Purification, Serological and Molecular Studies on an Egyptian Isolate of Faba Bean Necrotic yellows Virus (FBNYV) Infecting Faba Bean Plants

Faba bean necrotic yellows virus (FBNYV) was isolated from naturally infected faba bean plants in Egypt expressing severe chlorosis, necrotic, leaf rolling and stunting. The identification was based on tests by indirect ELISA, symptomatology, serological properties and polymerase chain reaction (PCR). Serological testing of 150 samples from faba bean plants showing virus- like symptoms and collected randomly from 3 governorate in Egypt showed that percentage of FBNYV natural infection was 17-23.6% from the growing seasons 2007-2009. The virus was purified by PEG and sucrose density -gradient centrifugation. The purified virus preparation had an ultraviolet absorption spectrum typical of the nucleoprotein with A₂₆₀/A₂₈₀ being 1.42. Yield of purified virus was 0.13mg/100g infected leaf tissue. Results of using polymerase chain reaction showed that, the simultaneous amplification of a 272 bp fragment of the FBNYV genome by using specific primer. The size of the PCR fragments was in agreement with expected for such nucleotide sequence. Serological and molecular methods, including enzyme-linked immunosorbent assay (ELISA), dot-blot immunoassay (DBIA), and immunocapture polymerase chain reaction (IC-PCR) were compared to evaluate their usefulness for diagnosis of this virus. Each method was tested with partially purified virus preparations and tissue samples from infected faba plants. Indirect ELISA was more sensitive than DBIA with all sample tested. Results showed that FBNYV was detectable in faba bean leaves up to 1:10^-5 dilutions, by indirect ELISA and up to l:10^-3 dilutions by DBIA. End-point dilutions of partially purified virus preparations from indirect and DBIA were 50 and 250 ng/ml, respectively. The most sensitive method was IC- PCR, in which the virus was detected in sap of infected leaves diluted to 1:10^-9 and 2ng/ml in partially purified virus preparation IC-PCR showed 16-fold and 32-fold greater sensitivity than indirect ELIAS and DBIA for detection of the FBNYV in partially purified FBNYV. Electron microscopy revealed virus particles were found scattered within the cytoplasm and occasionally in chloroplast and mitochondria which were highly deformed losing their structure.