Double-antibody Sandwich Enzyme-linked Immunosorbent Assay Research Articles

Cord Blood Ischemia-Modified Albumin Levels in Preterm Infants with Intrauterine Growth Restriction

Abstract Background Intrauterine growth restriction (IUGR) is one of the causes of perinatal mortality and morbidity. It is the failure of the fetus to achieve intrinsic growth potential. Since IUGR neonates are more likely to suffer complications including perinatal asphyxia, cold stress, hypoglycemia, hypocalcaemia, hyperbilirubinemia, feed intolerance, NEC, sepsis and even mortality. So, it is important that these infants are identified and managed appropriately at birth. Purpose The present study aims to determine the possible association between cord blood ischemia-modified albumin (IMA) levels and intrauterine growth restriction (IUGR) in preterm infants with or without complicated gestations. Patients and Method Eighty mothers and their preterm neonates (<37 weeks’ gestation) identified in 2 groups according to antenatal ultrasound: a group with prospective antenatal diagnosis of IUGR and the other group with normal fetal development, after consideration of inclusion and exclusion criteria. Cord blood Albumin and Ischemia modified albumin were estimated by double-antibody sandwich Enzyme-Linked Immunosorbent Assay (ELISA). Results Cord blood IMA was significantly higher in IUGR group compared to control group. Consequently, albumin level was significantly lower in IUGR group compared to other group. IMA was inversely correlation with Hb level and albumin level was inversely correlated with liver function tests (ALT and AST). Conclusion There is a positive association between IMA level and IUGR. This sent a message that combined antenatal ultrasound and serum IMA is a better predictor of intrauterine growth restriction and subsequent better care of neonates with IUGR.

Effects of shoot tip size on in vitro regeneration and virus elimination of grapevine cv. superior seedless

In this study, we investigate the relation between the explant size and the success of virus elimination in ‘Superior Seedless’ grape variety. Plants were tested by prior to thermotherapy and in vitro culture by using Double Antibody Sandwich-Enzyme-Linked Immunosorbent Assay (DAS-ELISA) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and verified as infected by different viruses (Grapevine leafroll associated virus -1+3, Grapevine leafroll associated virus -4 Strains, Grapevine rupestris stem pitting associated virus). Different size of shoot tip explants (0.1-0.5-1-5-10 mm) were excised and cultured on MS medium supplemented with 1 mgl-1 BA. All of the explants bigger than 0.1 mm 100% regenerated while 70% of the 0.1 mm explants showed complete regeneration and turned into a whole plant. Plants regenerated from 10 mm shoot tip explants have all three viruses (GLRaV-1+3, GLRaV-4 strains and GRSPaV) as founded in mother plant. Plants regenerated from 5 mm explants have two of them (GLRaV-1+3, GLRaV-4 strains) and plants regenerated from 1 mm explants have one (GLRaV-1+3). Plants regenerated from 0.1 mm explants were found completely virus-free.

A paper-based ELISA for rapid sensitive determination of anaphylaxis-related MRGPRX2 in human peripheral blood

Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH2 groups for covalent immobilization of the capture antibody. Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment.

Lipid Peroxidation Level and Peroxidase Activity in (Vitis vinifera L.) Leaves Infected with Grapevine Leafroll-Associated Virus 3

Objective: Grapevine (Vitis vinifera L.) is one of the major crops widely cultivated for the wine industry, as well as for the production of fresh and dried fruit in Azerbaijan. Grapevine leafroll disease (GLD) affects the vines throughout the world and is considered the most economically destructive among all virus and virus-like diseases. Vineyard surveys were conducted to determine the virus infection in the major grapevine growing region of Azerbaijan during 2019-2020. Methods/Statistical analysis: Forty-six samples were collected from grapevine fields and screened by rapid one-step assay AgriStrip and double-antibody sandwich Enzyme-linked immunosorbent assay (DASELISA). In our study, the levels of malondialdehyde (MDA), hydrogen peroxide (H2O2), pigments, relative water content (RWC), alterations in the activities of peroxidase enzymes (the activities of ascorbate peroxidase (APO), benzidine peroxidase (BPO) and guaiacol peroxidase (GPO) were investigated. Findings: The results revealed that tested samples were infected with Grapevine leafrollassociated virus 3 (GLRaV-3), however, no sample was found infected with other Grapevine leafroll-associated viruses. Changes in the levels of MDA, H2O2, pigments, RWC, alterations in the activities of peroxidase enzymes were studied. Significant reduction in green pigments like chlorophylls (a, b and total) and a gradual reduction in carotenoids were observed in all infected species. Obtained results showed that the level of RWC and the amount of hydrogen peroxide had increased in all infected leaves. The activities of APO, BPO and GPO were observed to increase in virus-infected leaves compared to the healthy control. Based on the results, it can be concluded that the effect of GLRaV-3 was more destructive in the N2 sample. The antioxidant defense system works more effectively in the N5 sample and this plant is more resistant to viral infection. Novelty: These results indicate that GLRaV-3 was the only most prevalent endemic viral pathogen of grapevine in the dominantly warmhumid continental climate of our country. All variable physiological parameters are assessed as the plant’s response to the pathogen. Keywords: activity; chlorophyll; hydrogen peroxide; malondialdehyde; peroxidase; virus disease

New Resources for the Specific and Sensitive Detection of the Emerging Tomato Brown Rugose Fruit Virus.

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

Development of Bottle Gourd Lines Resistant to Zucchini Yellow Mosaic Virus Using Ethyl Methanesulfonate Mutagenesis

Zucchini yellow mosaic virus (ZYMV) causes serious damage to cucurbit crops worldwide and can be spread by aphids, by mechanical injury, and in seeds. With the popularization of cucurbit grafting, the use of susceptible rootstock has increased the risk of ZYMV infection in cucurbit crops. In China, the bottle gourd (Lagenaria siceraria) is a widely used rootstock in grafted watermelon production. However, few resistant bottle gourds are available commercially. This study developed bottle gourd lines resistant to ZYMV using ethyl methanesulfonate (EMS) mutagenesis. A new mutated bottle gourd population (M1) was generated by treating seeds with EMS. Diverse phenotypes were observed in the seedlings, flowers, and fruit of M2 plants, some of which are of potential commercial interest, such as dwarfing and different fruit shapes. Based on the M2 phenotypes, 106 M3 lines were selected and screened for resistance to ZYMV by mechanical inoculation and agroinfiltration. Nine M3 lines were resistant to ZYMV during three tests. One inbred M4 line (177-8) was developed and showed stable resistance and no virus when tested using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and polymerase chain reaction. These resistant lines are promising materials for developing watermelon rootstock and exploring resistance genes as new ZYMV-resistant resources. EMS induction could be a practical strategy for creating resistant cucurbit crops.

Use of positive selection technique to manage yam viruses in seed yam production by Ghanaian farmers

Positive selection technique (PST) involves physical examination of plants at vegetative stage for viral disease symptoms and using tubers from apparently symptomless plants as planting materials in seed yam production. This study determined PST use among farmers in Atebubu-Amantin and Ejura-Sekyedumase Municipalities of Ghana. Viral diseases were observed on 63 PST and 63 non-PST farms within these Municipalities. PST farms (two in third season and two starters) were selected to assess the impact of continuous PST practice on plant establishment, disease incidence, severity and yield. Harvested seed yams from asymptomatic and symptomatic plants were sampled for virus detections using Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA). Viral disease incidence (35.70%) and severity (1.51) observed on PST farms were significantly lower than non-PST farms with 54.11% and 2.80, respectively (p< 0.05). PST farms in the third season had higher plant establishment (66 plants/30 m2) and yield (48.32 kg/30 m2) with lower disease incidence (28%) and severity (1.50) compared to PST starters which respectively were 60 plants/30 m2, 33.14 kg/30 m2, 52% and 2.3 (p< 0.05). DAS-ELISA showed that, 85% of harvested seed yams from symptomatic plants were infected with YMV, CMV, or YMMV as against 35% seed yams from asymptomatic plants. YMV was highly accumulated in seed yams from symptomatic plants (0.344) than those from asymptomatic plants (0.269). The current findings show that PST if practised by farmers could minimise viral diseases in seed yams. However, poor observation of viral symptoms by farmers and latent viral infection of plants could be limitations which need to be considered in further studies. Key words: Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA), latent viral infection, virus accumulation, yam viruses.

Association between Leptin and Adiponectin in women with Polycystic Ovary Syndrome in North Indian Population

Polycystic ovary syndrome (PCOS) is one of the most common endocrine abnormalities, characterized by biochemical hyperandrogenaemia, chronic anovulation, and polycystic ovaries. The objective of the present study to determine the association of adiponectin and leptin with women with PCOS in North Indian Women. In the hospital-based case-control study 100 women with PCOS and 100 controls were recuited from RAMA Medical College, Kanpur, India. Five ml venous blood sample was collected under aseptic precaution and transferred in the serum separator tubes. Leptin and adiponectin levels were assayed by human sensitive leptin double-antibody sandwich enzyme-linked immunosorbent one-step process assay (QAYEE-BIO life science) according to instructions provided with the kit. In the multivariable logistic regression analysis, leptin was found to be independently associated with 1.14 times higher risk for PCOS (OR 1.14, 95% CI 1.01 to 1.29). Receiver operating curve (ROC) analysis suggested excellent predictive accuracy of the multivariable model with Area under the curve 0.86. In the ROC analysis leptin was associated with 68% area under the curve for predicting PCOS using cut off value of &gt;7.87, (P&lt;0.001, Sensitivity 87.8, Specificity 49.4). Adiponectin level was not significantly associated with predicting PCOS (Area under the curve 0.54, P =0.30, Sensitivity 71.1, specificity 50.5, cut off value &lt;10.2). To conclude we observed that higher leptin levels were independently associated with the risk of PCOS, however adiponectin level was not independently associated with the risk of PCOS.

Development of double antibody sandwich ELISA as potential diagnostic tool for rapid detection of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus, a highly pathogenic viral agent is responsible for severe fatal hemorrhagic infections in many parts of the world. The early diagnosis of CCHF infection is important for successful clinical management and epidemiological control. The nucleoprotein (NP) of CCHFV being highly conserved and immunogenic is used as early diagnostic marker. In this study, we report a rapid and sensitive double antibody based antigen capture ELISA to detect Crimean-Congo hemorrhagic fever virus (CCHFV). Highly specific polyclonal and monoclonal antibody against NP has been generated and used as capture and detector antibody respectively. The assay was able to detect viral nucleoprotein in different matrices including human serum, ticks and culture supernatant. The detection limit of the developed sandwich ELISA assay was 25 ng of purified antigen. Comparison with a real time RT-PCR revealed its detection limit to be 1000 genome equivalents of CCHFV. Further the assay was comparatively evaluated with a commercial kit employing gamma irradiated CCHFV, revealing a sensitivity and specificity of 100%. This newly developed sandwich ELISA (sELISA) with high sensitivity and specificity could be used as an efficient method for the detection of CCHF virus in humans, ticks and culture supernatant. The assay will be useful as alternate tool for diagnosis of acute infection and is amenable for screening of large scale samples in resource limited settings.

Development and evaluation of indirect double-antibody sandwich ELISA for rapid detection of Salmonid Alphavirus using Baculoviridae expressed E1 Protein

Salmon alphavirus (SAV) is a widespread virus that infects Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) in Europe. It has a fatality rate of ≤48% and can cause pancreatic and sleeping disease. With the frequent exchange of salmon farming trade, it may become a global pathogen; therefore, a sensitive, effective, and high-throughput detection method is necessary. The serological diagnoses for SAV are indirect immunofluorescence assays (IFA) and indirect enzyme-linked immunosorbent assays (ELISA), which can only detect one of the six subtypes of SAV. In this study, an insect cell/baculovirus expression system was used for simultaneous detection of multiple genotypes (SAV1, SAV2, and SAV5) and an indirect double-antibody sandwich-enzyme linked immunosorbent assay (IDAS-ELISA) method was adopted, based on the highly conserved region (E1) of SAV1-6. The results showed that the IDAS-ELISA had no cross-reaction with other common RNA viruses and high sensitivity with 103.4TCID50/0.1 mL detection line. SAV 1 (98.33%) and SAV 2 (91.67%) samples were positively identified by IDAS-ELISA and real-time polymerase chain reaction (PCR), respectively. This assay can simultaneously detect multiple subtypes of SAV and provide technical support for clinical diagnosis and the epidemiology of SAV.

Novel Capsid-Specific Single-Domain Antibodies with Broad Foot-and-Mouth Disease Strain Recognition Reveal Differences in Antigenicity of Virions, Empty Capsids, and Virus-Like Particles

Foot-and-mouth disease (FMD) vaccine efficacy is mainly determined by the content of intact virions (146S) and empty capsids (75S). Both particles may dissociate into 12S subunits upon vaccine manufacturing, formulation, and storage, reducing vaccine potency. We report the isolation of capsid-specific llama single-domain antibodies (VHHs) with broad strain recognition that can be used to quantify intact capsids in FMD vaccines by double antibody sandwich (DAS) ELISA. One capsid-specific VHH displayed remarkably broad strain reactivity, recognizing 14 strains representing the 13 most important lineages of serotype A, and two VHHs cross-reacted with other serotypes. We additionally show that the newly isolated VHHs, as well as previously characterized VHHs, can be used to identify antigenic differences between authentic 146S and 75S capsids, as well as corresponding genetically engineered virus-like particles (VLPs). Our work underscores that VHHs are excellent tools for monitoring the quantity and stability of intact capsids during vaccine manufacturing, formulation, and storage, and additionally shows that VHHs can be used to predict the native-like structure of VLPs.

Different Methods for Detection and Diagnosis of Tomato Spotted Wilt Tospovirus Infection of some Cucurbit Host Plants and its Main Vector of Thrips Species in Field and Greenhouse in Egypt

The study recorded for the first time in Egypt the presence of tomato spotted wilt virus (TSWV) on cucurbits, which was confirmed using several serological tests, like double antibody sandwich ELISA (DAS-ELISA); Dot-blot ELISA assay and Immuno capture polymerase chain reaction (IC-PCR). By using Dot blot immunoassay test reaction against TSWV antisera, the ability of thrips species Thrips tabaci (Lind), Thrips palmi (Karny), and Frankliniella occidentalis (​Pergande), on the transmission of the (TSWV) were asserted. In the greenhouse, eleven plant species were determined as the host range of virus and field samples showed the similar symptoms. The mechanical transmission of TSWV showed a positive reaction bearing symptoms on the leaves of Cucumber, zucchini, smoke, datura and tomato plants 15-20 days after inoculation with transmission rates of 46.5, 40, 46.5, 46.5 and 86.5%, respectively. DAS-ELISA using antisera of TSWV showed that the greenhouse leaves samples were infected; otherwise, an amplification of IC-PCR products of 500 bp was detected by agarose electrophoresis of total RNA isolated from infected leaves.

A Double-Antibody Sandwich ELISA for Sensitive and Specific Detection of Swine Fibrinogen-Like Protein 1.

Fibrinogen-like protein 1 (FGL1), a member of the fibrinogen family, is a specific hepatocyte mitogen. Recently, it has been reported that FGL1 is the main inhibitory ligand of lymphocyte activating gene 3 (LAG3). Furthermore, the FGL1-LAG3 pathway has a synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway and is regarded as a promising immunotherapeutic target. However, swine FGL1 (sFGL1) has not been characterized and its detection method is lacking. In the study, the sFGL1 gene was amplified from the liver tissue of swine and then inserted into a prokaryotic expression vector, pQE-30. The recombinant plasmid pQE30-sFGL1 was transformed into JM109 competent cells. The recombinant sFGL1 was induced expression by isopropyl-β-d-thiogalactoside (IPTG) and the purified sFGL1 was used as an antigen to produce mouse monoclonal antibody (mAb) and rabbit polyclonal antibody (pAb). After identification, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for sensitive and specific detection of sFGL1 was developed. Swine FGL1 in samples was captured by anti‐sFGL1 mAb followed by detection with anti‐sFGL1 rabbit pAb and HRP-conjugated goat anti-rabbit IgG. The limit of detection of the developed sFLG1-DAS-ELISA is 35 pg/ml with recombinant sFLG1. Besides, it does not show cross‐reactivity with the control protein. Then serum samples of PRRSV-negative and -positive pigs were tested with the established DAS-ELISA and calculated according to the equation of y=0.0735x+0.0737. The results showed that PRRSV infection enhanced the serum FGL1 levels significantly. Our research provides a platform for the research on the functional roles of swine FGL1.

Grapevine Pinot gris virus infecting grapevines in Romania - Short Communicaiton

Grapevine Pinot gris virus (GPGV) has been identified in many grape growing countries of the world since 2012. The aim of this work was to investigate the presence of GPGV on some accessions collected from a germplasm collection, in addition to the propagation material and clonal selection samples. During 2019–2020, a total of 199 samples have been analysed by a double antibody sandwich – enzyme-linked immunosorbent assay (DAS-ELISA) for the presence of GPGV, Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus-1+3 (GLRaV-1+3) and Grapevine fleck virus (GFkV). Among them, 107 samples (53.76%) showed a GPGV-infection, associated with or without symptoms on the leaves (deformations, chlorosis, mosaic, wrinkles) or stunting plants. The distribution of infected varieties showed a high rate of infection in old varieties (37.38%), followed by clones (32.71%), rootstocks (11.21%), clonal selections (9.35%) and new varieties (9.35%). The tests revealed the association of GPGV with GFkV (5 cases) and GLRaV-1+3 (2 cases). GPGV should be included in the rules of grapevine certification schemes for the production of virus-free mother plants.

Identification, prevalence and genetic diversity study of potato viruses in Northeastern states of India

AbstractPotato affecting viruses is an important biotic factor that hampers both the quality and yield of potato production worldwide. The occurrence of potato viruses has been historically recorded in India. Viruses such as potato virus X (PVX), potato virus Y (PVY), potato virus A (PVA), potato virus M (PVM), potato virus S (PVS), potato leafroll virus (PLRV) have been recorded in all potato growing zones. To investigate the presence and distribution of potato virus diseases in North‐East India, a survey of seven major potato growing regions was conducted during the 2017 and 2018 growing seasons. The presence of potato viruses was confirmed by double antibody sandwich ELISA (DAS‐ELISA) with reverse transcriptase polymerase chain reaction (RT‐PCR) performed on selected samples. The sequencing followed by annotation of PCR amplicons obtained from RT‐PCR showed 95–100% nucleotide sequence identities with respective virus accessions on NCBI GenBank. The multiple sequence analysis and phylogenetic studies provided an overview of the genetic diversity pattern among different potato virus isolates. This study reveals a broad picture of the potato virus infections occurring in Northeastern states of India, which may be useful to develop effective control strategies against the potato viruses.

Protecting potato plants against PVX and PVY viral infections by the application of native and chemically modified legume proteins

Methylated soy and chickpea proteins (MSP and MCP, respectively), and 11S globulin (a soy protein fraction), are characterized by net positive charges and a more hydrophobic nature, nominating them as antiviral proteins. Under greenhouse conditions, potato plants (cv. Spunta) mechanically infected with potato virus Y (PVY) and potato virus X (PVX) were treated with MSP, MCP, and 11S globulin at different concentrations, after ten days of infection. The three tested substances inhibited the viral propagation, where the concentration 500 µg ml−1 produced the maximum antiviral action. This influence was further augmented by applying the material twice 10 and 20 days after the viral infection. Assessing the viral load by a double antibody sandwich ELISA (DAS-ELISA), dot-blot hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) confirmed the viral inhibition, in the following order: MSP > MCP > 11S globulin. Bringing the virus in contact with MCP produced deformed TEM (transmission electron microscope) viral micrographs referring to a potential direct action of the substance on the virus. The tested materials' antiviral activity at 500 µg ml−1 was positively reflected on the growth and yield of PVY or PVX-infected potato plants cultivated under open field conditions in January. Considerably high increases in the tuber yield of either PVY-infected (68, 42, and 36%) or PVX-infected plants (52, 41 and 30%), were produced by the treatment with MSP, MCP and 11S (500 µg ml−1), respectively.

Characterization of cucumber mosaic virus and its satellite RNAs associated with tomato lethal necrosis in Serbia

A four-year survey (2012–2015) was carried out to examine the genetic diversity of cucumber mosaic virus (CMV) isolates infecting tomato, as well as the presence and diversity of their satellite RNAs (satRNAs), collecting a total of 226 samples throughout the most important growing regions in Serbia. Besides CMV-like symptoms, the collected samples also exhibited more severe symptoms, such as systemic necrosis of leaves, branches and stems, accompanied by fruit malformation and necrosis. In a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), the presence of CMV was detected in approximately one quarter of the tested samples. Identification of CMV was confirmed by conventional reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of the coat protein (CP) gene of a group of 11 selected Serbian CMV isolates. Phylogenetic analysis of the CMV CP sequences of these selected isolates revealed their heterogeneity, as they fell into two different subgroups, IA and II. An additional RT-PCR analysis of CMV positive isolates using satellite RNAs specific primers detected the presence of satRNAs in eight samples. Sequence and phylogenetic analyses showed that Serbian CMV satRNAs variants were very heterogeneous, belonging to necrogenic and non-necrogenic variants. Necrogenic variants were divided into two groups, B and B1, containing a characteristic ‘necrogenic consensus’ sequence at the 3′ end of the RNA. A necrotic phenotype co-determined by satRNAs was expressed in inoculated tomato plants.

Molecular Characterization of the Alfalfa mosaic virus Infecting Solanum melongena in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid.

During the spring of 2019, distinct virus-like symptoms were observed in the Kafr El-Sheikh Governorate in Egypt in naturally infected eggplants. Leaves of affected plants showed interveinal leaf chlorosis, net yellow, chlorotic sectors, mottling, blisters, vein enation, necrotic intervention, and narrowing symptoms. The Alfalfa mosaic virus (AMV) was suspected of to be involved in this disease. Forty plant samples from symptomatic eggplants and 10 leaf samples with no symptoms were collected. The samples were tested by double antibody sandwich ELISA (DAS-ELISA) using AMV-IgG. Six of the 40 symptomatic leaf samples tested positive for AMV, while, DAS-ELISA found no AMV in the 10 leaf samples without symptoms. The AMV Egyptian isolate (AMV-Eggplant-EG) was biologically isolated from the six positive samples tested by DAS-ELISA and from the similar local lesions induced on Chenopodium amaranticolor and then re-inoculated in healthy Solanum melongena as a source of AMV-Eggplant-EG and confirmed by DAS-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) assay with a pair of primers specific for coat protein (CP) encoding RNA 3 of AMV yielded an amplicon of 666 bp from infected plants of Solanum melongena with AMV-Eggplant-EG. The amplified PCR product was cloned and sequenced. Analysis of the AMV-Eggplant-EG sequence revealed 666 nucleotides (nt) of the complete CP gene (translating 221 amino acid (aa) residues). Analysis of phylogeny for nt and deduced aa sequences of the CP gene using the maximum parsimony method clustered AMV-Eggplant-EG in the lineage of Egyptian isolates (shark-EG, mans-EG, CP2-EG, and FRE-EG) with a high bootstrap value of 88% and 92%, respectively. In addition to molecular studies, melatonin (MTL) and salicylic acid (SA) (100 μM) were used to increase the resistance of eggplant to AMV- infection. Foliar spray with MLT and SA caused a significant increase in the morphological criteria (shoot, root length, number of leaves, leaf area, and leaf biomass), chlorophyll and carotenoid content, antioxidant enzymes, and gene expression of some enzymes compared to the infected plants. On the other hand, treatment with MLT and SA reduced the oxidative damage caused by AMV through the reduction of hydrogen peroxide, superoxide anions, hydroxyl radicals, and malondialdehyde. In conclusion, MLT and SA are eco-friendly compounds and can be used as antiviral compounds.

Incidence and molecular characterization of potato leaf roll virus in seed potato production in Serbia

The distribution and frequency of potato leaf roll virus in the four most important potato growing regions in Serbia were studied during the seven years (2012–18). One hundred randomly collected potato tubers were sampled from each seed lot. The young leaves that developed in three weeks were sampled and tested to record infection rate. The presence of potato leaf roll virus was detected by double-antibody sandwich enzyme-linked immunosorbent assay and disease incidence was calculated using standard formula. The obtained result showed that the highest prevalence of potato leaf roll virus was detected from seed potato samples originated from the Raski region during 2018 (20.7%), while in the Moravicki region, only 2.3–11.1% of the potato leaf roll virus was detected every year. The average annual potato leaf roll virus infection was the highest in 2012 (8.4%) and 2018 (8.0%). For further confirmation of potato leaf roll virus infection, reverse transcription (RT)-PCR was performed using specific primers PLRVCPvEcoRI /PLRVCPcNcoI, designed to amplify a 650 bp fragment of the full-length coat protein gene. The PCR products derived from 26 isolates were directly sequenced using the same primer pair as in RT-PCR. The coat protein sequence analysis revealed that the Serbian potato leaf roll virus isolates showed very low nucleotide diversity (95.9–100%). They shared the highest nt identities of 98.08–99.36% with the sequences of potato leaf roll virus isolates deposited in the GenBank from other parts of the world. Phylogenetic analysis and the haplotype network of the coat protein gene sequences showed that the Serbian potato leaf roll virus isolates could be classified in two different groups indicating two possible introductions of the virus to Serbia. The results of this study confirmed the importance of potato leaf roll virus in seed potato production in Serbia. Additionally, this research highlights the need for a continuous monitoring of the potato seeds produced in Serbia as well as imported seeds for the presence of potato leaf roll virus.

Development of a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for the detection of KHV.

Koi herpesvirus disease (KHVD) caused by the koi herpesvirus (KHV) is difficult to diagnose in live fish, presenting a challenge to the koi industry. The enzyme-linked immunosorbent assay (ELISA) method cannot be widely used to detect KHV because few commercial anti-KHV antibody exists. Here, we developed an anti-ORF132 polyclonal antibody and confirmed its reactivity via indirect immunofluorescence assay and Western blotting. A double-antibody sandwich ELISA (DAS-ELISA) was established to detect KHV, monoclonal antibody 1B71B4 against ORF92 was used as the capture antibody, and the detection antibody was the polyclonal antibody against the truncated ORF132. The lowest limit was 1.56ng/ml KHV. Furthermore, the DAS-ELISA reacted with KHV isolates, while no cross-reactions occurred with carp oedema virus, spring viraemia of carp virus, frog virus 3 and grass carp reovirus. Two hundred koi serum samples from Guangdong, China, were used in the DAS-ELISA test, and the positive rate of the koi sera was 13%. The clinical sensitivity and specificity of the DAS-ELISA relative to the traditional PCR method were 66.7% and 97.6%, respectively. Our findings may be useful for diagnosing and preventing KHVD in koi and common carp.