Dilution End Point Research Articles

Biological Exploration and Physicochemical Characteristics of Tomato Brown Rugose Fruit Virus in Several Host Crops

Tomato brown rugose fruit virus (ToBRFV) is considered an emerging disease and a viral pandemic for tomato consumers. The objectives of this research were to analyze the biological and physicochemical characteristics of ToBRFV in tomato and tobacco plants, as well as to evaluate its natural host range. Inoculant seeds were recovered from ToBRFV-infected tomato samples in Coahuila, Mexico, and confirmed by RT-PCR. In the first greenhouse experiment, tomato plants of the F1 hybrid variety 172–300 (Yüksel), infected with ToBRFV, were used to evaluate viral inclusions (VI), dilution endpoint (DEP), the incubation period (IP), and latency period (LP). In a greenhouse experiment, Nicotiana longiflora plants were inoculated with ToBRFV to determine the in vitro longevity (IVL) and thermal inactivation (TI) of the virus in sap. Finally, the inoculation of tomato plants grown in open fields was carried out to evaluate transmission to natural hosts. The plants tested for possible ToBRFV reservoirs near the inoculum source were inspected and confirmed by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The results indicate that the VIs on tomato leaves manifested as X-bodies and rounded, stacked plaques within epidermal cells. The DEP required to induce the infection in plants was from a ToBRFV concentration of 1 × 10−5.5, the IP of ToBRFV occurred 9 to 12 days post-inoculation, and LP could be detected one day after inoculation. The TI of ToBRFV in N. longiflora plants occurred at 85 °C for 30 min. Ipomoea purpurea, Mirabilis jalapa, Clematis drummondii, and Solanum tuberosum were newly identified hosts of ToBRFV. The results found contribute to a better understanding of the impact of ToBRFV, managing and preventing the spread of ToBRFV in diverse environments.

Use of Pseudomonas protegens to Control Root Rot Disease Caused by Boeremia exigua var. exigua in Industrial Chicory (Cichorium intybus var. sativum Bisch.).

Boeremia exigua var. exigua is a recurrent pathogen causing root rot in industrial chicory. Currently, there is no chemical or varietal control for this disease, and thus, management strategies need to be developed. This study determined the biocontrol effect of strains of Pseudomonas protegens bacteria with antimicrobial compounds on the fungus B. exigua var. exigua under in vitro, in vivo, and field conditions. In addition, root colonization by these bacteria was estimated by the phlD-specific PCR-based dilution end point assay. Eighteen isolates of Pseudomonas spp were evaluated, and the strains that showed the greatest in vitro inhibition of fungal mycelial growth (mm), Ca10A and ChB7, were selected. Inoculation with the strain ChB7 showed less severity (necrotic area) under in vivo conditions (root trials) compared with the control inoculated with the pathogen (p ≤ 0.05). The molecular analysis revealed that the root colonization of plants grown in pots was equal to or greater than 70%. Similar levels were observed in the field trials conducted at the Selva Negra and Canteras experimental stations (2015-2016 season), with values ranging from 85.7 to 70.5% and from 75.0 to 79.5%, respectively. Regarding yield (ton ha-1), values were higher in the treatments inoculated with strains Ca10A and ChB7 (p ≤ 0.05) at both experimental sites, while a lower incidence and severity of root rot were observed at Selva Negra. These results suggest that the Chilean strains of P. protegens are a promising tool for the control of root diseases in industrial chicory.

Uji Infektivitas Tobacco Mosaic Virus (TMV) dan Cucumber Mosaic Virus (CMV) pada Chaenopodium amaranticolor

The infectivity test is one of the completeness in knowing the physical properties of the virus. Determination of the physical properties of the virus can be done by: Thermal inactivation point (TIP), in vitro resistance (Longevity in vitro / LIV), and dilution end point (DEP). The purpose of this study was to determine the infectivity of TMV and CMV in sap through LIV, DEP, and TIP.LIV testing is done by sap incubation until the test time (0 hours, 6 hours, and 24 hours), DEP test is done by diluting sap (not diluted, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7), while TIP test is done by heating sap at room temperature, 50oC, 70oC, 90oC for 10 minutes. The results of this study show TMV is stable compared to CMV.TMV is still infectious in sap storage for 24 hours, while CMV is not infectious between 6-24 hours after incubation. The cutoff point of sap TMV dilution is between 10-5 and 10-6, while CMV has been unable to cause symptoms in indicator plants at dilutions between 10-1 and 10-2. TMV is still infectious at 90oC temperature treatment, while CMV inactivation hot spots are 40oC and 50oC.

Development of the NRTL functional activity coefficient (NRTL-FAC) model using high quality and critically evaluated phase equilibria data. 2

One of the most significant models based on the local composition concept in advanced thermodynamics, is the non-random two-liquid (NRTL) model. Many studies have been conducted to enhance its applicability to various systems. Among these studies, the development of the NRTL model for electrolytic systems, polymer systems, and systems containing special chemical associations has been particularly significant. However, the NRTL model and its developed forms are based on the concept of local composition, and the dependency of these models on the experimental data for regressing NRTL interaction parameters limits their applicability of the NRTL model. Accordingly, to overcome this limitation, the NRTL functional activity coefficient model (NRTL-FAC), which is a group contribution model, was developed (https://doi.org/10.1016/j.fluid.2021.113088). The current study aims to extend the NRTL-FAC model. To achieve this, 15 new main groups have been added to the previous ones, and 266 new interaction parameters have been optimized. A total of 374 isothermal/isobaric VLE data sets, including 7115 data points, were used. The quality of the experimental VLE data was assessed using various thermodynamic consistency tests, including the Herington test, Van Ness test, point test, infinite dilution test, EoS test, and endpoint test. The NRTL-FAC model can accurately predict the VLE of binary systems, with results in good agreement with experimental data at different temperature and pressure ranges. This accuracy was observed for both azeotrope systems and ideal systems with positive or negative deviation from Raoult's law. Unlike the UNIFAC-DMD model, which restricts a portion of its interaction parameters to consortium members, the NRTL-FAC model interaction parameters are publicly accessible. Therefore, the NRTL-FAC model can be confidently used as a preliminary estimate to predict VLE behavior in process design when experimental data is not available.

Physical Properties of Potyvirus on Chilli (Capsicum annuum) of Doon Valley in Uttarakhand

A virus is transmitted mechanically in the plant host. Potyviridae is a family of Potyvirus,consisting of a positive-sense RNA genome and filamentous particles. It has a single typeof protein coat. Mostly, this virus is identified in Leguminosae and Solanaceae family. Theplant virus is transmitted by mechanical inoculation and aphids. Chilli, cucurbits, potatoes,peas, tomatoes, and other plants were infected with the bean yellow mosaic virus, pea mosaicvirus, bean yellow mosaic virus, sugarcane mosaic virus, and peanut mosaic virus. Thermalinactivation point, dilution endpoint, longevity in vitro, and longevity in vivo were used toidentify the physical properties of a potyvirus. Several isolates mild and severe were collectedin the chilli plant. The infected plant developed necrotic leaf spots and chlorotic symptoms.

Multiple Levels of Triggered Factors and the Obligated Requirement of Cell-to-Cell Movement in the Mutation Repair of Cucumber Mosaic Virus with Defects in the tRNA-like Structure.

Simple SummaryBased on analysis of the tRNA-like structure (TLS) mutation in cucumber mosaic virus (CMV), mutation repair is correlated with several levels of triggered factors, including the dose of inoculation of virus mutants, the quantity effect on corresponding viral RNA, and the quality effect on corresponding viral RNA. All types of TLS mutation in different RNAs of CMV can be repaired at a low dose around the dilution end-point. At a high dose of inoculation, TLS mutations in RNA2 and RNA3, but not RNA1, can be repaired, which correlates with the relative quantity defect of RNA2 or the genome size defect of RNA3. In addition, all the above types of mutation repair necessarily require cell-to-cell movement, which presents the obligated effect of cell-to-cell movement on mutation repair.Some debilitating mutations in RNA viruses are repairable; however, the triggering factors of mutation repair remain largely unknown. In this study, multiple triggering factors of mutation repair are identified based on genetic damage to the TLS in CMV. TLS mutations in different RNAs distinctively impact viral pathogenicity and present different types of mutation repair. RNA2 relative reduction level or RNA3 sequence change resulting from TLS mutation is correlated with a high rate of mutation repair, and the TLS mutation of RNA1 fails to be repaired at the high inoculum dose. However, the TLS mutation of RNA1 can be repaired at a low dose of inoculation, particularly around the dilution end-point or in the mixed inoculation with RNA2 having a pre-termination mutation of the 2b gene, an RNAi suppressor. Taken together, TLS mutations resulting in quality or quantity defects of the viral genome or TLS mutations at low doses around the dilution end-point are likely to be repaired. Different levels of TLS mutation repair necessarily require cell-to-cell movement, therefore implying its obligated effect on the evolution of low-fitness viruses and providing a new insight into Muller’s ratchet. This study provides important information on virus evolution and the application of mild viral vaccines.

Development of the NRTL functional activity coefficient (NRTL-FAC) model using high quality and critically evaluated phase equilibria data. 1

The non-randomness two-liquid model (NRTL) has been used successfully in chemical engineering to calculate phase equilibria. Different forms of the NRTL model have been developed, including original NRTL, electrolyte NRTL (e-NRTL), polymer NRTL and segment based NRTL (NRTL-SAC). The mentioned forms of the NRTL model are correlative so that they require experimental data to regress the NRTL interaction parameters, which in turn, limit the application of the NRTL models. In the current study, therefore, the NRTL functional activity coefficient (NRTL-FAC) model was developed based on the high-quality experimental vapor-liquid equilibrium (VLE) data. The quality of experimental data were checked using different thermodynamic consistency tests, including Herington test, Van Ness test, point test, infinite dilution test, EOS test and endpoint test. Of the available VLE data, 2938 isothermal/isobaric data sets including 46,651 data points were used in the development of the model. Besides, 137 molecules with 50 main groups were used to develop the NRTL-FAC model. The Stavermann-Guggenheim term was also used to account the combinatorial contribution of the NRTL-FAC model. The good agreement between the predicted results by the NRTL-FAC and the experimental VLE data is encouraging. The present model performs superior to the previous versions of the UNIFAC model.

Biological and Physico-chemical Properties of Causal Agent of Virus Disease of Coccinia barteri (Hook. f.) Keay in Calabar, Cross River State

The physico-chemical properties of the causal agent of virus disease of Coccinia barteri (Hook. f.) Keay were studied. The virus causing the disease was characterized using diagnostic tools such as host range, longevity in vitro, thermal inactivation point, dilution endpoint and aphid transmission. The virus was mechanically transmitted from the natural host (C. barteri) to the healthy test plants in the green house. In the biological properties, the virus was successfully transmitted by Aphis spiraecola (obtained from Chromolaena odorata (L.) R. M. King & H. Rob.) from infected Cucumeropsis mannii Naudin to a healthy C. mannii in a non-persistent manner and had a narrow host range limited to the family Cucurbitaceae. In the physico-chemical properties based on crude sap with an unknown virus concentration, beyond which infectivity was lost. It was readily inactivated by heating to 35 – 65°C for 10 minutes in determination of thermal inactivation point. The virus had a longevity in vitro of between 4 – 5 days beyond which it was non-infectious. Symptoms induced by the virus were leaf cupping, mottle chlorosis, blisters, stunted growth, rugosity, leaf malformation and mosaic patterns.

Cloning and Analysis of Authentic Patient-Derived HCV E1/E2 Glycoproteins.

Experimental characterization of the properties of authentic viruses circulating in infected individuals presents a problem when investigating RNA viruses with error-prone polymerases. The hepatitis C virus provides an extreme example of RNA virus genetic variability, as the nucleotide composition of HCV genomes can vary by more than 30% between strains. The envelope glycoproteins E1 and E2 in particular are able to tolerate a particularly high level of variation. They are under continual selection pressure from the host antibody response during chronic infection and can tolerate adaptive mutations, leading to great diversity in a single host. The diversity of E1/E2 in circulating viruses has hindered investigations of their function and development of a vaccine that will generate antibodies able to potently neutralize entry of genetically distinct strains.Here we describe methods used in our laboratory to overcome the limitations of investigating the properties of the envelope glycoproteins representing only small numbers of HCV variants. Using a high-fidelity, limiting dilution ("endpoint") PCR approach to amplify single E1/E2 cDNA templates, which can then generate recombinant model viral particles using retrovirus packaging/reporter constructs. These retroviral pseudoparticles (pseudotypes) facilitate investigation of the properties of authentic E1/E2 glycoproteins in a single-round infection assay. We also describe optimized methods for generation of infectious pseudoparticles from patient-isolated E1/E2 and methods for performing neutralization assays with both anti-virus and anti-host antibodies.

Biological and molecular evidence of papaya ring spot virus pathotype P from mid-hills of Meghalaya, India

Papaya ring spot virus (PRSV) is the limiting factor of papaya production in India irrespective of agro-climatic conditions and it may cause crop losses up to 85–90%. In this study, diagnostic survey on PRSV was conducted in mid-hills of Ri-Bhoi district, Meghalaya, India during April to June, 2015. Reverse transcription polymerase chain reaction assay detected PRSV infection in 38 of 53 samples collected from surveyed locations with an average incidence of 71.7%. The representative isolate (PRSV-P:Umiam) from mid-hill Meghalaya was maintained in healthy papaya seedlings by standard sap inoculation method and typical symptoms were expressed within 21–24 days post inoculation. Studies on the physical properties revealed that the dilution end point of PRSV-P:Umiam at 10−2, longevity in vitro for 18 h and thermal inactivation point in between 50 and 55 °C. Among the cucurbitaceous hosts, pumpkin (Cucurbita pepo) alone exhibited chlorotic lesions on leaves within 20–25 days post inoculation. The partial coat protein (CP 750 bp) of PRSV-P:Umiam gene shared maximum nucleotide (~ 95%) and amino acid (~ 98%) identity with PRSV isolates reported from northern and eastern India. The CP based phylogeny clustered PRSV-P:Umiam with the PRSV isolates from north and east India.

Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter.

The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA. Graphical abstract The roots of metrological traceability for human nuclear DNA mass concentration results. Values for the factors in blue must be established experimentally. Values for the factors in red have been established from authoritative source materials. HHGE stands for "haploid human genome equivalent"; there are two HHGE per diploid human genome.

Isolation and characterization of basil mosaic virus in Egypt

Different viral infection symptoms were observed on leaves of basil plants (Ocimum basilicum L.), growing at El-Monofia Governorate, Egypt, during 2015–2016. The majority of these symptoms were mosaic and leaf malformation. The pathogenic agent of this disease is known as basil mosaic virus (BsMV). The sensitive hosts to BsMV are Chenopodium amaranticolor and Phaseolus vulgaris cv. (brown beans). The biological properties of BsMV were found to be 10−4, 6 days and 66 °C for dilution end point, longevity in vitro and thermal inactivation point, respectively. The electron micrograph of the isolated virus indicated this virus was found to be isometric particles of approximately, 50 nm in diameter and could be classified as tentative member of the Fabavirus. Real-time PCR detection by the Fab primer pairs was confirmed at Ct value = 25.44 for the total cDNA isolated from infected Basil plants and 27.94 for the cDNA of purified virus particles, while CMV primer pairs showed no amplification for both samples. According to the available reports this seems to be the first record for basil mosaic virus in Egypt.

Biological and Molecular Studies on an Egyptian Potyvirus Isolate from Hyocyamus muticus L.

ANEW isolate of potyvirus was isolated from naturally-infected……. henbane plants (Hyocyamus muticus L.) collected from thefarm of Faculty of Pharmacy, Cairo University in January 2013. Thenaturally infected henbane plants exhibited severe mosaic, rugosity,blistering and malformation. Thermal inactivation point, dilution endpoint and longevity in vitro of virus isolate were found to be 65 oC,10- 4 and 4 days, respectively. Electron micrograph of partial purifiedvirus negat ively s t ained wit h 2% p hosp hot unges t ic acidrevealed the presence of filamentous viruses with size 1440 X14.3nm.Total RNA was extracted from infected henbane plant. Comparativenucleotide sequence analysis for virus showed a high degree ofsimilarity (62 %) with four potyviruses accessions (EU482153: Potatovirus Y isolate Foggia, KF850513: Potato virus Y isolate M3,AM184113: Henbane mosaic virus partial gene for polyprotein-PHYS/H isolate, AY166867: Potato virus Y strain N isolate). Thevirus isolate was published in GenBank with accession numberKM497011.

Importance of early detection of vancomycin-resistant subpopulations in apparently susceptible meticillin-resistant Staphylococcus aureus clinical isolates.

This study aimed to investigate the presence of vancomycin-non-susceptible subpopulations in apparently susceptible meticillin-resistant Staphylococcus aureus (MRSA) and the ability of these isolates to develop into homogeneously resistant strains. Vancomycin MICs of 200 clinical MRSA isolates were determined using agar dilution (AD) and spiral gradient endpoint (SGE) technique. Isolates with an MIC≤2mg/L but displaying subpopulations with an MIC>2mg/L by SGE were re-tested by Etest and PAP-AUC and were incubated with 2mg/L vancomycin for 2 weeks. MIC testing was repeated weekly by AD, Etest and SGE to observe progression to non-susceptibility. A total of 17.5% and 16.0% of isolates were non-susceptible to vancomycin (MIC>2mg/L) by SGE and AD, respectively. Eight isolates (4%) displayed a resistant subpopulation; five met the definition of hVISA by PAP-AUC. The initial Etest MIC for these isolates was 2mg/L, but resistant subpopulations were observed in only three isolates on prolonged incubation. MICs of all eight isolates increased rapidly in the presence of vancomycin, reaching ≥3.0mg/L by Day 7 and ≥4mg/L after 14 days by all three methods. The prevalence of vancomycin-non-susceptible MRSA was high, and non-susceptibility developed rapidly in seemingly susceptible isolates with covert subpopulations. These were effectively detected by SGE. With increasing reports of vancomycin clinical failure, early detection of potentially non-susceptible isolates before or early in vancomycin therapy is essential to avoid further resistance development and poor clinical outcomes. SGE offers a novel and cost-effective technique for detection of potentially non-susceptible strains.

Bahasa Indonesia

Mosaic disease in yard long bean is caused by Bean common mosaic potyvirus (BCMV) and has been reported to affect yield. Common method to detect infection of BCMV involves serological assay and polymerase chain reaction (PCR). The aims of this research is to assess the sensitivity of three methods, i.e. Indirect Enzym-Linked Immunosorbent Assay (I-ELISA), Dot Immunobinding Assay (DIBA), and reverse transcription (RT)-PCR as detection method for BCMV infection in yard long bean. Sensitivity level of the methods was evaluated by diluting plant extract and antisera for I-ELISA and DIBA, and cDNA as template in RT-PCR. Virus isolate from Cirebon was maintained in yard long bean in screenhouse and used for the assessment. Absorbance value of ELISA showed that dilution end point for I-ELISA was reached at 10 -3 and 10 -2 of plant extract and antisera dilution, respectively. Positive infection was still detected using DIBA when the plant extract was diluted up to 10 -5 based on development of color intensity on nitrocellulose membrane. Specific viral DNA fragment was still amplified when cDNA was diluted up to 10 -4 , indicated higher sensitivity level of RT-PCR method.

Characterization and Confirmation of Papaya ringspot virus-W Strain Infecting Trichosanthese cucumerina at Tamil Nadu, India

Mosaic and leaf blisters symptoms were observed in Trichosanthese cucumerina (Snake gourd) in regions of coimbatore, India. The biological, physical, serological and molecular properties of the virus isolate were studied. Virus isolate produce chlorotic local lesions on Chenopodium amaranticolor and systemic symptoms on all inoculated cucurbitaceous hosts. The particles appear flexuous rod shape of 750×12 nm under electron microscope. Serologically this isolate identified as strain of PRSV. Dilution end point of virus isolate was 10-4. Thermal inactivation point and Longevity in-vitro of the virus isolate was found to be 55°C and 30 h respectively.

Quantitative Molecular Detection of Xanthomonas hortorum pv. carotae in Carrot Seed Before and After Hot-Water Treatment.

Molecular assays to detect and quantify DNA from viable cells of the seedborne pathogen Xanthomonas hortorum pv. carotae in carrot seed were developed and evaluated for use on nontreated and hot-water-treated seed lots. Both a TaqMan real-time polymerase chain reaction (PCR) assay and a loop-mediated isothermal amplification (LAMP) dilution endpoint assay detected and quantified DNA from viable pathogen cells after treatment of carrot seed washes with the live-dead discriminating dye propidium monoazide (PMA). The detection limits of the assays were approximately 101 CFU for pure cultures of X. hortorum pv. carotae, and 102 to 103 CFU/g seed from naturally infested carrot seed lots. X. hortorum pv. carotae in and on carrot seed was killed by soaking the seed in hot water (52°C for 25 min), and a subsequent PMA treatment of these hot-water-treated seed washes suppressed detection of the pathogen with both the real-time PCR and LAMP assays. For 36 commercial seed lots treated with PMA but not hot water, regression of colony counts of X. hortorum pv. carotae measured by dilution plating on a semiselective agar medium versus estimates of pathogen CFU determined by the molecular assays resulted in significant (P ≤ 0.05) linear relationships (R2 = 0.68 for the real-time PCR assay and 0.79 for the LAMP assay). The molecular assays provided quantitative estimates of X. hortorum pv. carotae infestations in carrot seed lots in <24 h, which is a significant improvement over the 7 to 14 days required to obtain results from the traditional dilution-plating assay.

First Report of Lettuce mosaic virus Infecting Lactuca sativa in India.

In January 2012, lettuce (Lactuca sativa L.) plants (19 out of 38) of one of the accessions (EC687345, variety NVRS-10:001818) exhibiting mild mosaic and stunted growth symptoms were observed at the Indian Agricultural Research Institute (IARI) experimental farm, New Delhi. Similar disease symptoms in lettuce plants in India were previously described (3) and the associated virus was characterized for host range, dilution end point, thermal inactivation point, and longevity in vitro. In this study, definitive molecular evidence is presented for the presence of Lettuce mosaic virus (LMV) infecting lettuce in India. Analysis of preparations from leaves of symptomatic samples with an electron microscope revealed flexuous virus particles measuring 750 × 13 nm, suggesting the association of a potyvirus (4). To identify the potyvirus infecting these lettuce plants, the 3' terminal portion of the genome including the part of the nuclear inclusion b (NIb), complete coat protein (CP) region, and 3' untranslated region (UTR) was amplified by RT-PCR, cloned, and sequenced. Total RNA was extracted from infected leaves using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to RT-PCR using potyvirus specific forward (5' ACCACAGGATCCGGBAAYAAYAGYGGDCARCC 3') and reverse (5' CACGGATCCCGGG(T17)V 3') primers (2). PCR products (~1.8 kb) were cloned into pGEM-T Easy vector (Promega, Madison, WI) and sequenced (GenBank Accession No. JQ794776). Sequence comparisons revealed the CP of the virus infecting lettuce (834 bp) shared 96 to 100% nucleotide and deduced amino acid sequence identity with the corresponding regions of LMV isolates AJ306288 and AJ297630 from the United Kingdom, CAA46603 and NC003605 from the United States, AJ278854 and AJ278854 from Brazil, and AJ488153 from China, thus complying with the cut off range of 90 to 99% for identifying isolates/strains of the same virus (1). Similarly, 99 to 100% nucleotide sequence identity was observed with the corresponding region of the 3'UTR (245 bp) while 93 to 96% nucleotide identity of NIb region (654 bp) with LMV isolates. These results confirm that the virus infecting the symptomatic lettuce plants was an isolate of LMV. The amino acid sequences (DAG and WCIEN) conserved among majority of potyviruses were also present. Since the virus is aphid transmissible, its natural infection on other hosts and spread can't be ruled out.

Molecular characterization of a cucumber mosaic cucumovirus isolated from lettuce in Egypt

Cucumber mosaic cucumovirus (CMV) was isolated from lettuce plants (Lactuca sativa) showing virus like symptoms. Isolation was performed depending on specific polyclonal antibodies and Chenopodium quinoa as a local lesion host. Virus was purified from 200gm of virus-infected Nicotiana tabacum cv. White Burley leaves giving A260/280 ratio of 1.21 and a yield of 1.7mg. Purified virus preparation was used for rabbit immunization to produce specific polyclonal antibodies. IgGs were purified and evaluated by indirect enzyme linked immunosorbant assay (I-ELISA) to determine the dilution end point which found to be 1:512. Electron micrographs showed spherical virus particles of about 30nm in diameter. Virus coat protein (CP) molecular weight was determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), giving a single band of 25kDa within resolving gel. Immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) was used for the amplification of CMV coat protein gene (cp), the appearance of 657bp bands confirmed the expected size of such gene. Comparing virus cp gene sequence with the sequences of seven overseas isolates confirmed that the under study isolate was related to the CMV subgroup I.

A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia

A suspected virus disease was identified from an arborescent Brugmansia x candida Pers. (syn. Datura candida Pers.) tree. The causal agent was aphid transmissible at low rates. Viral particles were purified from infected tobacco tissue, analyzed, and purified virions were inoculated into healthy tobacco plants to recreate the symptoms. The virions had a mean length of 720-729 nm, and infected cells contained inclusion bodies typical of potyvirus infections. Analysis of infected tissues and purified virions with a panel of potyvirus-specific antibodies confirmed identification as a potyvirus. Viral host range, dilution end point, thermal tolerance and aphid transmission characteristics were examined. The viral genome (9761 nt) is typical of potyviruses, with the closest related potyvirus being pepper mottle virus, at 72 % nt sequence identity. Based on conventions for naming novel potyviruses, the virus was determined to be a member of a previously undescribed species, tentatively named "Brugmansia mosaic virus" (BruMV).