Serological and molecular characterisations of the Egyptian isolate of Bean common mosaic virus

Bean common mosaic virus (BCMV) was isolated from the naturally infected bean plants collected from the Kafr El-Sheikh and El-Gharbia Governorates. BCMV induced sever mosaic, vein banding, malformation, leaf curling and stunting on bean plants cv. Giza 6. The isolated virus was propagated in bean plants cv. Giza 6. The identification of BCMV was carried out serologically by an indirect enzyme-linked immunosorbent assay using BCMV antiserum. Positive reaction indicated that the virus under study was related serologically to Potyvirus. The molecular biology techniques were used to identify and characterise the coat protein gene of BCMV. Oligonucleotide primers were designed for BCMV according to the published nucleotide sequences of BCMV and were successfully amplified with a DNA fragment (300 bp) from BCMV CP gene by RT-PCR. The total RNA was extracted from bean leaves and was reverse-transcribed and amplified using the oligonucleotide primer. The amplified product was analysed by gel electrophoresis. Also, Southern and dot blot hybridisations were used to establish the authenticity and specificity to the RT-PCR-amplified products of BCMV. The nucleotide sequences of the Egyptian isolate of BCMV/CP showed similarity with an isolate (BCMV-NY 15) which belongs to Puerto Rico.     

Immunocapture RT-PCR detection of Bean common mosaic virus and strain blackeye cowpea mosaic in common bean and black gram in India

The strains of Bean common mosaic virus (BCMV) and blackeye cowpea mosaic (BICM), genus Potyvirus, were detected from 25 common bean and 14 black gram seeds among 142 seed samples collected from different legume-growing regions of India. The samples were subjected to a growing-on test, an indicator plant test, an electron microscopic observations, an enzyme linked immunosorbent assay and an immunocapture RT-PCR. The incidence of the two tested viruses in common bean and black gram seed samples was 1–6% and 0.5–3.5%, respectively in growing-on test evaluations. Electron microscopic observations revealed filamentous virion particles from the leaves of plants showing characteristic virus disease symptoms in growing-on and host inoculation tests. The identity of the strains was confirmed by immunocapture RT-PCR, with a final amplification product of approximately 700 bp for BCMV and BCMV–BICM. The complete identity of the two viruses was further confirmed by nucleotide sequencing of the partial coat protein and 3′-UTR regions. The sequences of the four BCMV and BCMV–BICM isolates each consisted of 583–622 and 550–577 nucleotides. The present report confirms the widespread nature of these two serious potyviruses in the two most important legume crops in India.     

Mutation spectrum of the PAH gene in the PKU patients from Khorasan Razavi province of Iran

Background

Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients.

Methods

Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11.

Results

A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G > A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11 + 1G > C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410).

Conclusion

With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen.     

Detection of cucumber mosaic virus in some ornamental plants and elimination of nonspecific ELISA reactions

During surveys carried out in 2008 in the nurseries of some ornamental and medical plants, about 90 plant samples belonging to six plant species were collected. Cucumber mosaic virus (CMV) was detected by routinely double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) in most tested plants. In Vinca rosea, Ocimum basilicum and Pelargonium sp., which reacted positively for CMV, 100% of the samples were infected. High absorbance values were obtained when extracts were prepared from these species and then examined by DAS-ELISA. The results indicated that CMV concentration on O. basilicum is greater than 50 μg ml^?1 when compared with purified CMV standards. High absorbance values occur even under conditions fully unsuitable for the detection of antigens. This result suggested that the strong ELISA absorbance values were nonspecific reactions with materials in plant extracts. So, other detection methods including dot blot, Western blot and bioassay was used for comparing with ELISA test. The nonspecific reactions were substantiated when CMV was not detected by dot blot and Western blot. Also, infectious CMV was not detected in bioassay involving inoculation of extracts onto Chenopodium amaranticolor and Nicotiana tabacum var. White Burley plants as indicator hosts. Addition of bovine serum albumin (BSA) or polyvinylpyrolidene (PVP) to extraction buffer or to wells after IgG coating for DAS-ELISA reduced nonspecific reactions. Finally, CMV was isolated from V. rosea symptomatic plants, which give a positive reaction by DAS-ELISA, dot blot, Western blot and bioassay. CMV vinca-isolate was identified based on transmission, host range, serological tests, purification and electron microscope.     

Serological and molecular characteristics of pea seed borne mosaic virus-PSbMV from lentil (Lens culinaris Medik L.) in Iran

Abstract

During 2016 growing season, samples with leaf yellowing and mosaic like symptoms were collected from main lentil (Lens culinaris Medik L.) fields. Specific ELISA positive PSbMV samples were selected for RT-PCR. Using specific pair of primer towards CP gene region of the virus, PCR product of approx.235?bp was amplified. Based on the nucleotide sequences of the CP gene PSbMV isolates were classified in two major groups. In which, isolates in group I divided into two subgroups of Iranian isolates (B2) and (G1), along with Czech Republic, Australia, China, Greece, Pakistan and Egypt were placed in the subgroup-I. Isolate (V18) from Iran placed independently in group II. In the grouping based on the amino acid sequencing the isolates divided into two phylogenetic groups. Iranian isolates along with an isolate from Australia categorized in group-II, however world isolates were all clustered in group-I.     

Incidence and severity of bean common mosaic disease and resistance of popular bean cultivars to the disease in western Kenya

The common bean (Phaseolus vulgaris) is a high protein crop and the main legume in the cropping system of western Kenya. Despite its importance, common bean yields are low (<1.0 t/ha) and declining. Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) are the most common and most destructive viruses and can cause a yield loss as high as 100%. In Kenya, a limited number of cultivars and exotic genotypes with resistance to BCMV and BCMNV strains have been reported. This study sought to determine the distribution and screen popular cultivars for resistance to the viruses. In October 2016 and May 2017, two diagnostic surveys for bean common mosaic disease (BCMD) were conducted in seven counties of western Kenya namely Bungoma, Busia, Homa Bay, Nandi, Vihiga, Kakamega and Siaya. Leaf samples showing virus-like symptoms were collected and analysed by ELISA. Sixteen popularly grown bean cultivars together with cowpea (Vigna unguiculata), soybean (Glycine max), green grams (Vigna radiata) and groundnut (Arachis hypogaea) were planted in a greenhouse in a completely randomized block design with three replicates. The plants were inoculated with BCMNV isolate at 3-leaf stage. Data were taken weekly for 3 weeks on type of symptoms expressed and number of plants infected. In total, 270 bean farms were visited. Symptoms of mosaic, downward curling, local lesions, stunting or a combination of these were observed during both surveys. Mean virus incidence was higher in the short rain season (50.2%) than in the long rain season (35.6%). The mean BCMD severity on a scale of 0–3 was highest (2.3) in Kakamega County and lowest (0.5) in Siaya. On variety resistance tests to BCMNV isolate, 10 bean cultivars were susceptible, four tolerant and two resistant. BCMNV is widely distributed across counties probably because of use of uncertified seeds by farmers and inoculum pressure from seed and aphid vector. For improved yields of common bean, farmers should be advised to plant certified seeds for all legumes in the cropping system.     

Biological and molecular characterization of lettuce mosaic virus from Tehran province in Iran

In this study, lettuce samples having LMV infection symptoms were collected from Tehran fields during 2003. Samples tested for LMV infection by immuno printing. Three positive samples in immuno printing collected and their characteristics were determined. In mechanical inoculation, these Isolates produced symptoms on Chenopodium quinoa, C. amaranticolor, Gomphrena globosa, Nicotiana benthamiana, Lactuca sativa cv. Mantilia and cv. Terocadero (which contains the mol1 resistance gene and susceptible respectively), but not cv. Salinas 88 (which contains the mol2 resistance gene). LMV was purified and LMV polyclonal antiserum was produced in rabbit by a series of intravenous and intramuscular injections, the precipitin titre of this antiserum was 1:1024. Gel double diffusion test (GDDT) was performed, and precipitin bands appeared. SDS-PAGE and western blotting showed the presence of coat protein 29 kDa. In IC-RT-PCR with on LMV specific primer pair, an approximately 1300 bp fragment was amplified.     

Serological comparison of an Australian isolate of capsicum mosaic virus with capsicum tobamovirus isolates from Europe and America

A New South Wales isolate (Ca) of capsicum mosaic virus was tested against antisera to it and capsicum tobamovirus isolates from the Netherlands (P8, P11), USA (SL), Argentina (FO) and Sicily (PM). The comparison demonstrated that the four viruses Ca, P8, PM and SL are closely related to each other, forming a series of decreasing relationship to Ca in the above order. FO was related to these but insufficiently to be considered part of the group, and P11 was only slightly related to the others. The literature on serology of tobamoviruses in Capsicum spp. was collated and it is suggested that isolates from Sicily (pepper mild mottle), Australia (capsicum mosaic), The Netherlands (P8, P14) and USA (SL) be considered as strains of a virus distinct from both tobacco mosaic and tomato mosaic viruses and that these isolates all be referred to in future as strains of pepper mild mottle virus.     

薇甘菊水杨酸羧甲基转移酶基因的分离鉴定及表达分析

为研究水杨酸羧甲基转移酶基因在植物防御系统中的作用,采用RACE法从薇甘菊(Mikania micrantha)cDNA文库中克隆到薇甘菊水杨酸羧甲基转移酶基因SAMT全长cDNA,并进行外源表达以及水杨酸诱导模式分析.结果表明,该基因cDNA全长1 299 bp,其中编码区长1 089 bp,编码362个氨基酸,Blast显示该基因编码的氨基酸序列与仙女扇(Clarkia breweri)的相似性为74%,证实其主要功能是将水杨酸(salicylic acid,SA)甲基化成水杨酸甲酯(methyl salicylate,MESA),由此将该基因命名为MmSAMT,GenBank登录号为FJ869889.将MmSAMT编码序列克隆至pET-32a(+)载体,转化Rosetta-gami(DE3)中表达,SDS-PAGE显示单体分子量在40 kD左右,与预测结果一致.Western bolt显示在20℃、0.05 mmol/LIPTG和180 r min-1下诱导6 h,可获得较多的可溶性蛋白质.对喷施100μmol/L SA薇甘菊叶片中MmSAMT的转录谱进行研究,该基因的诱导受到SA的激活,48 h的表达水平达最高,暗示MmSAMT可能通过催化合成MeSA引发系统获得性抗性,提高抗性防御的警戒等级. 89.将MmSAMT编码序列克隆至pET-32a(+)载体,转化Rosetta-gami(DE3)中表达,SDS-PAGE显示单体分子量在40 kD左右,与预测结果一致.Western bbt显示在20℃、0.05 mmol/LIPTG和180 r min-1下诱导6 h,可获得较多的可溶性蛋白质.对喷施100μmol/L SA薇甘菊叶片中MmSAMT的转录谱进行研究,该基因的诱导受到SA的激活,48 h的表达水 达最高,暗示MmSAMT可能通过催化合成MeSA引发系统获得性抗性,提高抗性防御的警戒等级. 89.将MmSAMT编码     

Southern and dot blot analysis of DNA from formalin-fixed,paraffin-embedded tissue samples from colonic carcinomas

Two extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffinembedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA. Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.     

Improved purification procedure and some serological and physica properties of cassava common mosaic virus from South America

Large quantities of cassava common mosaic virus (CCMV) were purified from systemically infected Nicotiana benthamiana plants. A polyclonal antiserum, with a titre of 1/128 in the tube precipitin test, was produced by immunising rabbits with purified virus. Viral antigens were detected in cassava, using both the double-antibody sandwich or plate-trapped antigen forms of enzyme-linked immunosorbent assay (ELISA). The virus reacted with antisera to the potexviruses potato virus X and tulip virus X in F(ab')₂ ELISA. As determined by ELISA, isolates of CCMV from cassava and chaya are closely serologically related to each other. Leaf extracts from infected N. benthamiana plants were infective to a dilution of 10^--⁴ but not 10^--⁵; after heating for 10 min at 65 °C but not 70 °C; and after storage at room temperature for 14 days. The virus has a sedimentation coefficient of 126 S_20,w, a single coat protein molecule of c . mol. wt 21 000, and a single-stranded RNA genome of c . mol. wt 2.0 ± 10⁶. Several dsRNA species, including the putative viral replicative form of c . mol. wt 4.1 ± 10⁶, were isolated from virus-infected cassava and N. benthamiana .     

Establishment and characterization of a new epithelial cell line, KC-1, from koala (Phascolarctos cinereus) conjunctiva

Summary A novel, untransformed koala cell line (KC-1) was established by culturing koala conjunctival tissue in growth medium, which has permitted the study of the cell biology of this unique system. After the establishment of the KC-1 cell line, the cells were characterized by light microscopy, doubling time, and Western blot analysis. Light microscopy revealed that the cells have an epithelial morphology. Doubling times were significantly different (P<0.015) depending on fetal calf serum (FCS) concentration (16.5 h in 10% FCS and 26.5 h in 2% FCS). Cells constricted while in suspension but were shown to attach to the coverslip (or flask) and flatten rapidly, less than 1 h after seeding. To confirm the epithelial nature of the cells, protein was extracted and Western blot analysis was performed. Subsequent probing with primary and secondary antibodies (monoclonal anticytokeratin clone C-11 IgG1 and anti-mouse IgG) revealed two bands at 45 and 52 kDa (compared against a protein molecular weight marker) that correspond to primary type I keratin and major type II keratin, respectively, expressed in simple epithelial cells. The koala cell line was adapted to grow continuously in Dulbecco modified Eagle medium containing 10% FCS for at least 30 passages. This unique cell line is an ideal tool for further investigatio on koala cell biology and cytogenetics and for exploration of the pathophysiological mechanism of eye infections caused by different pathogens in koalas.     

Epidemiology and molecular typing of Candida isolates from burn patients

This study, spread over a span of 2 years describes Candida infections in burn patients of an Indian hospital. A total of 220 burn patients were monitored and Candida could be isolated from 138 patients. A total of 228 different Candida species were obtained from various body locations of these patients. Species identification revealed that Candida albicans was the most predominant (45) followed by Candida tropicalis(33), Candida glabrata (13.5), C. parapsilosis (4), C. krusei (2.75) and C. kefyr (1.75). DNA fingerprinting of all C. albicans isolates was done by using CARE-2 probe. Fingerprinting analyses of all the C. albicans strains revealed that strains collected from different patients were different. It is noteworthy that patients with disseminated candidiasis had a similar, but unique strain isolated from all body locations, suggesting a possibility that commensal isolates might be turning pathogenic. Taken together, this is probably the first ever detailed survey of Candidainfections in burn patients in India and is expected to lead to better clinical management of this group of patients.     

Schistosoma mansoni: molecular characterization of Alkaline Phosphatase and expression patterns across life cycle stages

Here we describe the cloning and characterization of the Schistosoma mansoni Alkaline Phosphatase (SmAP), previously identified in the tegument of adult worms. SmAP encodes a complete sequence composed of 536 amino acids containing an N-terminal signal peptide, five N-glycosylation sites, and a GPI anchor signal, similar to that described for mammalian orthologs. Real-time RT-PCR and Western blot experiments suggest a rapid translation as soon as cercariae are transformed into schistosomula. Immunolocalization analysis shows that the protein is widely distributed in the worm tissues, with increased concentration in the vitelline glands of female parasites. Furthermore, the surface localization of this enzyme was quantitatively supported by its enzymatic activity in live ex vivo or cultured parasites throughout the life cycle stages. The fact that cercariae accumulate large amounts of SmAP mRNA, which rapidly translates into protein upon schistosomula transformation, indicates it may have an important role in host invasion.     

Phospholipid molecular species distributions of Candida isolates from the UK and Iran

AIMS: Some species of Candida have been shown to differ with respect to their polar lipid fingerprints when analysed by fast atom bombardment mass spectrometry (FABMS). The aims of this study were to contribute to the existing body of information by (i) examining representatives of species not previously examined and (ii) seeking strains differences associated with country of origin (UK or Iran). METHODS AND RESULTS: FABMS analysis was performed on extracted lipids of 22 strains representing eight species of Candida. The most abundant anion (19 isolates) in spectra was with mass to charge (m/z) 281, corresponding to C18:1 carboxylate. The major phospholipid analogue anions were m/z 515 and 501 (13 strains). These anions were putatively identified as the phosphatidyl molecular species PA(23 : 2) and PA(22 : 2) respectively. Data for strain pairs were compared using the Pearson's coefficient of linear correlation. The values generated were used to cluster strains by nearest-neighbour linkage, using both carboxylate and phospholipid analogue anion data. Isolates of C. parapsilosis were clearly distinct from other isolates. Iranian isolates tended to cluster together when phospholipid anion data were used. However, if carboxylate anion data were used, four Iranian isolates of C. albicans were tightly clustered with three UK isolates, of which two were C. albicans and one was C. dubliniensis. CONCLUSION: It is concluded that both lower, and higher, mass peaks in FABMS spectra can be of potential value in comparing Candida isolates from different countries and from different species. SIGNIFICANCE AND IMPACT OF THE STUDY: When polar lipids of different Candida species are compared, it is important to bear in mind that geographical differences affect results as has been observed with bacteria in similar studies.     

Detection of Prunus necrotic ring spot virus in plum,cherry and almond by serological and molecular techniques from India

Stone fruits are cultivated in the temperate and sub-temperate regions of India. During surveys in stone fruit growing areas, viral symptoms were observed in almond, cherry and plum. These samples were brought to the laboratory for further detection at serological and molecular levels to check the presence of virus. In the present study, incidence of PNRSV is reported on plum (Prunus domestica), almond (Prunus dulcis) and cherry (Prunus avium) using serological and molecular techniques. Coat protein gene of PNRSV was amplified from almond, cherry and plum. This is the first molecular evidence of PNRSV on these stone fruits reported from India.