A simple real-time polymerase chain reaction assay using SYBR Green for hepatitis C virus genotyping.

Detection of the hepatitis C virus (HCV) genome is crucial for diagnosis of HCV infection and for monitoring the efficacy of HCV treatment. Thus, we aimed to develop a convenient screening test for common HCV genotypes based on melting curve analysis with PCR. Serum samples were drawn from 124 patients with known HCV infection confirmed to be antibody and HCV RNA positive. A characteristic melting curve was obtained by monitoring the fluorescence as the temperature increased through the melting point of the PCR product. Results were compared with those obtained by the restriction fragment length polymorphism (RFLP) genotyping method. The melting curve analysis indicated that the different genotypes had discrete melting points (P < 0.0001): 90.43 +/- 0.065 degrees for genotype 1 (n = 35), 90.21 +/- 0.064 degrees for genotype 2 (n = 18), 90.62 +/- 0.045 degrees for genotype 3 (n = 29) and 90.84 +/- 0.130 degrees for genotype 4 (n = 42). The genotype was determined for all samples using the newly developed method as well as RFLP, and the two systems produced concordant results. The sensitivity of the assay was 91.4 % for genotype 1, 83.3 % for genotype 2, 93.1 % for genotype 3, and 85.7 % for genotype 4. Genotypes detected by melting curve analysis significantly correlated with those detected by RFLP (r = 0.946, P < 0.0001) with a strong linear relationship (r 2 = 0.895). This melting curve analysis is a rapid, convenient and low-cost screening test for differentiation of HCV genotypes 1-4.

Occurrence of iris yellow spot virus from onion crops in Egypt.

Iris yellow spot virus (IYSV) is a infects onion bulb and seed crops in many countries including Egypt. Results of the mechanical inoculation reveled that, small chlorotic lesions and systemic necrosis were observed on both Nicotiana benthamiana and Datura stramonium after 10 days, while there were no symptoms were appeared on the onion plant. The viral biological transmission with Thrips tabaci was highly reported to be efficiently for virus transmitted. Our results confirmed the presentence of virus-like particles of a Tospovirus infected onion leaf using transmission electron microscopy. Both of sequence and phylogenetic analysis of N gene revealed that our viral isolate is IYSV with 95 % identity with reported Israel isolate. The sequence of N gene had three motifs: casein kinase II Phosphorylation site, N-myristoylation site and protein kinase C phosphorylation site. These motifs are involved in regulation, activity and stability of IYSV. To our knowledge, this is the first molecular characterization of IYSV in Egypt.

First Report of Iris yellow spot virus Infection of Garlic and Egyptian Leek in Egypt.

Egyptian leek (Allium ampeloprasum), garlic (A. sativum), and onion (A. cepa) are key vegetables produced by small- and large-scale farmers in Egypt for national and international markets. Iris yellow spot virus (IYSV; family Bunyaviridae, genus Tospovirus) is an economically important viral pathogen of bulb and seed onion crops in many onion-growing areas of the world (1,3). During February and March of 2011, symptoms of spindle-shaped, straw-colored, irregular lesions with occasional green islands were observed on onion, garlic, and Egyptian leek cultivated on large and small farms in Dakahlia, Gharbia, Kalubia, Menofia, Qena, and Assiut governorates in Egypt. The presence of IYSV was confirmed by specific double antibody sandwich (DAS)-ELISA Flash Kits (Agdia Inc., Elkhart, IN) (2). A survey was carried out by collecting 100 plant samples (10 asymptomatic and 90 symptomatic) of each plant species from fields in the governorates of Dakahlia, Gharbia, Kalubia, Menofia, Qena, and Assiute and testing the plants using DAS-ELISA. For onion and garlic, 45% of the symptomatic samples and 0% of the asymptomatic plants tested positive. For leek, 34% of the symptomatic samples tested positive and 0% of the asymptomatic samples. ELISA-positive samples were tested using a reverse transcription (RT)-PCR assay with primers specific to the S RNA of IYSV (forward primer 5'-TAAAACAAACATTCAAACAA-3' and reverse primer 5'-CTCTTAAACACATTTAACAAGCAC-3') (2). Amplicons of approximately 1,100 bp were obtained from all symptomatic samples that were ELISA positive, but none of the asymptomatic plants nor the sterile water control sample produced PCR amplicons. The amplicons were cloned (at least three clones per plant species) using the TOPO TA Cloning Kit (Invitrogen, Grand Island, NY), and sequenced. The Egyptian onion IYSV isolate (GenBank No. JN541273) had the greatest nucleotide sequence identity (86%) with the corresponding S RNA region of IYSV isolates from India (GenBank Nos. EU310290, EU310284, and EU310276). The Egyptian garlic IYSV isolate (GenBank No. JN541275) showed the strongest identity (93%) with that of a Sri Lankan IYSV isolate (GenBank No. GU901211). The Egyptian leek IYSV isolate (GenBank No. JN541274) exhibited 91% sequence identity with that of the same Sri Lankan isolate (No. GU901211). To our knowledge, this is the first report of IYSV infecting garlic and Egyptian leek in Egypt. IYSV infection of onion was reported previously from the agricultural farm of the Faculty of Agriculture, Cairo University, Giza (4), but to our knowledge, this is the first report of natural infection by the virus in commercial onion production in Egypt. Further surveys and monitoring of IYSV incidence and distribution in the entire Egyptian governorate are under investigation. References: (1) D. H. Gent et al. Plant Dis. 88:446, 2004. (2) H. R. Pappu et al. Arch. Virol. 151:1015, 2006. (3) H. R. Pappu et al. Virus Res. 141:219, 2009. (4) A. Manal et al. Egypt. J. Virol. 3:49, 2006.