Occurrence, Distribution and Relative Importance of Viruses Infecting Hot Pepper and Tomato in the Major Growing Areas of Ethiopia

Hot pepper and tomato fields in the main growing areas in the Rift Valley and the west of Ethiopia were surveyed for virus infections in 1994. A total of 286 samples from hot pepper and 222 samples from tomato plants and associated Datura stramonium L. and Nicandra physalodes Gaertn. weeds with symptoms suggestive of virus infections were collected and analysed using electron microscopy, serology and test plant reactions. Potato virus Y (PVY), Ethiopian pepper mottle virus (EPMV), pepper veinal mottle virus (PVMV) and tomato mosaic virus (ToMV) were detected in hot pepper samples while tomato samples were shown to be infected with tomato mild mottle virus (TMMV), PVY and ToMV. The most widespread and predominant viruses which also occurred frequently in mixed infections were PVY and EPMV in hot pepper and PVY and TMMV in tomato. TMMV was also found in many samples of D. stramonium and N. physalodes. ToMV was identified in only few samples from both crops in the Rift Valley by its characteristic particle morphology, serological properties and symptomatology. PVMV was found in hot pepper samples only from western Ethiopia, but no natural infection of tomato with this virus was revealed. This is the first report on the natural occurrence of TMMV in tomato, D. stramonium and N. physalodes, as well as of ToMV in hot pepper and tomato in Ethiopia.     

Incidence and epidemiology of Pepper veinal mottle virus (PVMV), a Potyvirus disease of pepper

There have been various reports about the devastating effect of Pepper veinal mottle virus (PVMV), a Potyvirus disease of pepper in Nigeria contributing to its low yield and reduced fruit quality leading to great economic loss. Different strains of the virus have been identified and characterised over the years and the disease incidence, severity and aphid vector distribution across agro-ecological zones studied. Different cultural management techniques have been tried and found to be effective with varying degree of success, and these included the use of organic manures, intercropping with tall companion crops, time and date of planting and the use of tolerant/resistant varieties. Integrated pest management techniques for PVMV disease have been found to be very effective.     

Evaluation of maize/pepper intercropping model in the management of pepper veinal mottle virus,genus Potyvirus,family Potyviridae on cultivated pepper (Capsicum annuum L.) in Nigeria

Three pepper cultivars obtained from National Horticultural Research Institute (NIHORT) Idi-Ishin, Ibadan were intercropped with maize for two planting seasons between March and September in each year. These pepper cultivars were NHV1-D96, and NHV1-E96 and NHV1-F96. A 90-day maturing maize variety (DMSR-1) was used as the intercropping companion plant. The pepper seedlings were raised in a greenhouse. A randomised complete block design was used for this experiment. Each variety was intercropped with maize and replicated three times including the sole plot. The results obtained for each year were not significantly different from each other. There was a significant difference in pepper veinal mottle virus (PVMV) disease incidence and severity at a probability of less than 5% in the treatment used. PVMV disease incidence and severity was relatively higher in the sole pepper crop compared with pepper intercropped with maize. In the three varieties of peppers intercropped with maize, less than 17% disease incidence and 15% disease severity were recorded in all the varieties with a minimum yield of 4 tons per hectare compared with the sole pepper cropping of the same variety that recorded as high as 75% disease incidence and 72% disease severity with a maximum yield of 3.3 tons per hectare. There was a significant negative correlation at probability less than 0.05 between disease incidence, severity and the fruit yield of pepper. Variety NHV1-F96 in the maize intercrop recorded the highest yield of 15.99 tons/ha with a land equivalent ratio of 2.4 tons/ha. The success of the PVMV disease management evaluated in this study was judged by the extent of reduction in number of diseased plants and by an increase in vigor of the cultivated pepper crop, with an increase in fruit yield and quality. This signifies that for devising effective viral disease management for any crop it is important that the vectors of the virus present in the particular area are exactly controlled from having contact with the target plant. The reduction of pest incidence with intercropping of non-host plants should be carefully considered.     

Studies on plant growth-regulating substances LV. The Plant growth-promoting properties of some β-aryl-propionic acids

Severe diseases of pepper (Capsicum annuum), tomato (Lycopersicon esculentum), eggplant (Solanum melongena) and tomato eggplant (Solanum integrifolium) in West Africa were induced by pepper veinal mottle virus (PVMV). Five selected virus isolates were serologically similar and readily transmissible by aphids in the non-persistent manner, but they differed in host range and/or symptoms induced in some susceptible species. One isolate from eggplant failed to infect pepper, Chenopodium quinoa and C. amaranticolor, and induced only local infections in tomato. An isolate from tomato failed to infect eggplant, and an isolate from tomato eggplant induced severe stunting in Physalis floridana. The type strain, like the isolate from tomato, failed to infect Nicotiana tabacum systemically, but each caused severe systemic leaf and stem necrosis in tomato. None of the isolates infected S. melongena cv. Long Purple, suggesting that PVMV might be controlled in this and perhaps other crop species by the use of immune or tolerant cultivars. All five isolates were serologically related to potato virus Y and some to six of 12 other potyviruses.     

Characterization of the monoclonal antibody against classical swine fever virus glycoprotein E<Superscript>rns</Superscript> and its application to an indirect sandwich ELISA

Classical swine fever virus (CSFV) E^rns is an envelope glycoprotein possessing RNase activity. The E^rns-based enzyme-linked immunosorbent assay (ELISA) has been considered a discriminating diagnostic test for differentiating infected from vaccinated animals. The purpose of this study was to produce a specific monoclonal antibody (MAb) to E^rns for further developing an indirect sandwich ELISA. The MAb CW813 was shown to specifically recognize both the monomer and dimer forms of Pichia pastoris yeast-expressed E^rns (yE^rns). The antigenic site recognized by MAb CW813 was mapped to the region of amino acid residues 101–160 of E^rns where it was neither a neutralizing epitope nor essential to RNase activity. Furthermore, MAb CW813 was utilized as a capture antibody to develop a yE^rns-based indirect sandwich ELISA for detecting swine antibody to E^rns. The assay demonstrated a high sensitivity and specificity that may provide an alternative method for developing a diagnostic kit with easy manipulation and low cost.     

Bell Pepper Mottle Virus, a Distinct Tobamovirus Infecting Pepper

Bell pepper mottle virus (BPeMV) can be distinguished by symptomatology and host range from other tobamoviruses but a reliable identification needs serological tests. The relationships of BPeMV to tobacco mosaic virus (TMV), Odontoglossum ringspot virus (ORSV), tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (PMMV) were investigated using precipitin drop tests on slides, immunodiffusion gel tests, double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and indirectELISA using enzyme-linked goat anti-rabbit globulins for the determination of antiserum titers and serological differentiation indices (SDI). Comparisons of SDIs and amino acid composition data demonstrated that BPeMV is a new species of the tobamovirusgroup. BPeMV, ORSV, PMMV, and TMGMV form a cluster within the genus (group) and could be considered as a subgenus of tobamoviruses.     

Monoclonal Antibody-Based Dipstick Assay: A Reliable Field Applicable Technique for Diagnosis of Schistosoma mansoni Infection Using Human Serum and Urine Samples

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.     

Systemic Infection of Tobacco by Pepper Veinal Mottle Potyvirus (PVMV) Depends on the Presence of Potato Virus Y (PVY)

In single inoculations, both PVY and PVMV replicated in inoculated leaves of Nicotiana tabacum cv. ‘Xanthi nc’ plants, but only PVY infected the tobacco plants systemically, whereas PVMV caused localized infection. A mixed infection by the PVY-To72 and PVMV-type strains was experimentally realized in ‘Xanthi nc’ plants. In the presence of PVY, PVMV migrated systemically into the upper leaves of the tobacco plant, as was proved by back inoculation. It would appear that in tobacco, PVY acts as a “helper” virus, providing PVMV with the necessary component factor for migration. In extracts from the co–infected leaves. Immune Electron Microscopy (IEM) revealed phenotypic mixed particles which contained a mixture of coat proteins of PVY and PVMV. The role of the structural and functional interactions between the two viruses, which enable PVMV to migrate systemically in tobacco plants, is discussed.     

Differentiation of Bulgarian Isolates from Cucumber Mosaic Virus by Serological Methods

Cucumber mosaic virus (CMV) is of great importance to the Bulgarian economy and hence a detailed knowledge of its diversity under local geographic and climatic conditions is required. An extended study was carried out on CMV strains the currently occur in Bulgaria. Fifty-one isolates and strains found in different regions and various crops were biologically characterized and serologically differentiated into subgroups I and II using different variants of enzyme-linked immunosorbent assay (ELISA) [double antibody sandwich (DAS)-, antigen-coated plate (ACP)-, triple antibody sandwich (TAS)- with poly and monoclonal antibodies] and immunodiffusion tests. The ELISA modifications with monoclonal antibodies individually (ACP) or in combination with polyclonal antibodies (TAS-ELISA) are suitable for mass screening of CMV isolates. The hyperimmune sera against strains from CMV subgroups I and II were very efficient for use in isolate differentiation via gel double immunodiffusion. The results obtained correlated with the polymerase chain reaction and restriction fragment length polymorphism data reported by other authors. The majority of the isolates belonged to subgroup I, whereas 10, mainly from tomato and pepper, belonged to subgroup II. Most of the subgroup II isolates came from the north of Bulgaria. The results of the present study will help to clarify the virus epidemiology and to develop specific control measures.     

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

Background

Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.

Methods

In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.

Results

The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).

Conclusions

These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.     

Naturally acquired IgM antibody response to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax in Korea: use for serodiagnosis of vivax malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-na?ve healthy individuals (control group 1), and 70 malaria-na?ve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.     

Improved methods for the diagnosis of African trypanosomosis

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered.     

Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library

The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, are time-consuming, and require appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage display vector pMECS with diversity of 2.8 × 10⁹. Based on phage display technology and periplasmic extraction ELISA, H7N2-specific VHH antibodies were successfully isolated. According to a pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo, and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin–streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/ml. Given the above, the newly developed VHH antibody-based double sandwich ELISA (DAS–ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus.     

One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30 min

This article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly sensitive detection of human C-reactive protein (CRP) in less than 30 min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimal process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads in 15 min, followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3 to 81 ng ml⁻¹ with a limit of detection of 0.4 ng ml⁻¹ and an analytical sensitivity of 0.7 ng ml⁻¹. It detects CRP spiked in diluted human whole blood and serum with high analytical precision, as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.     

双抗体夹心ELISA与NIH法在狂犬病疫苗抗原检测中的比较

通过用单克隆抗体制备双抗体夹心ELISA,快速检测狂犬病疫苗中狂犬病毒糖蛋白(G)的含量,将狂犬病疫 苗的检定时间由28天缩短至2个工作日,以此缩短疫苗库存待检时间,提高疫苗的生产和销售效率,并最终替代 NIH动物法。将待检的狂犬病疫苗样品在同一性别12～14g昆明鼠体内作效力检定试验(NIH),同时用双抗体夹 心ELISA检测狂犬病疫苗中狂犬病毒G蛋白含量,用Microsoft Office Excel做出标准品和各样品疫苗的线性关 系图并计算出疫苗效力值E-NIH。结果用双抗体夹心ELISA所得的E-NIH与对应的小鼠效力试验所得结果M- NIH之间呈正相关性;同一批狂犬病疫苗分次测得E-NIH值在2．41～5．85之间,而相应的M-NIH值在5．11～ 10．19之间。从而得出E-NIH与相应的M-NIH之间存在明显的线性关系;与NIH法比较,ELISA具有重复性好、 成本低、快速等优点;用双抗体夹心ELISA替代小鼠效力试验是可能并可行的。     

Comparison of Vero cell assay, polymerase chain reaction and an enzyme immunoassay for identification of verocytotoxin-producing Escherichia coli O157:H7

This study evaluated three different analytical methods for identification of Verocytotoxin-producing E. coli O157:H7 (VTEC) strains. A total of 34 E. coli O157:H7 strains isolated from bovine faeces and bovine carcasses were comparatively tested with Vero cell assay (VCA), PCR and the sandwich ELISA "RIDASCREEN Verotoxin" test. The VCA, performed without a neutralization assay, gave a false positive result because a VCA-positive E. coli O157:H7 strain did not possess the VT-coding genes when tested with PCR. The lack of specificity of the VCA could be avoided by testing for neutralization of cytotoxicity. The commercial ELISA system was as sensitive and specific as PCR, with the advantages of being a more rapid and easier procedure which could be employed in all first level diagnostic laboratories.     

Sensitive ELISA detection of amyloid-beta protofibrils in biological samples

Amyloid-beta (Abeta) protofibrils are known intermediates of the in vitro Abeta aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Abeta protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Abeta protofibril immunoassay, Abeta conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Abeta protofibrils without interference from Abeta monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Abeta protofibrils in both cell and animal models, proving that Abeta protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Abeta protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Abeta protofibrils in AD and has the potential of becoming an important diagnostic assay.     

Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin

Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.