Triple Antibody Sandwich ELISA Research Articles

A SNP mutation affects rhizomania-virus content of sugar beets grown on resistance-breaking soils

Rhizomania is one of the most devastating biotic stresses affecting sugar beet (Beta vulgaris L.). It is caused by Beet necrotic yellow vein virus (BNYVV) vectored by the plasmodiophorid Polymyxa betae K. The only means available to control the disease is the use of genetically resistant varieties. “Rizor” or “Holly” (Rz1) and WB42 (Rz2) have been the most widely used resistance sources in the commercial varieties. Recently, naturally occurring resistance-breaking (RB) rhizomania strains have been identified causing major concerns. The aim of this study was to identify SNP mutations that show associations with resistance to rhizomania in sugar beet plants grown under resistance-breaking (RB)-BNYVV soils. Rhizomania virus content was evaluated by indirect triple-antibody sandwich-ELISA within two F 2 segregating populations respectively grown on an AYPR and IV-BNYVV strain infected soils. Bulked segregant analysis (BSA) was performed. The resistant and susceptible plants were genotyped with a 384-SNPs panel. Of the 384 SNPs, SNP249 was found to associate with the resistance both to the AYPR strain (R 2 = 0.37; P = 0.0004) and to the IV-BNYVV (R 2 = 0.09; P = 0.0074). Our results suggested that the SNP249 could be readily applicable for marker-assisted breeding of resistance to AYPR strain of rhizomania.

Characterization of Blueberry shock virus, an Emerging Ilarvirus in Cranberry.

Blueberry shock virus (BlShV), an Ilarvirus sp. reported only on blueberry, was associated with scarring, disfigurement, and premature reddening of cranberry fruit. BlShV was detected by triple-antibody sandwich enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction, and isometric virions of 25 to 28 nm were observed in cranberry sap. The virus was systemic, although unevenly distributed in plants. The coat protein of BlShV from cranberry shared 90% identity compared with BlShV accessions from blueberry on GenBank. Phylogenetic analysis of isolates of BlShV from cranberry collected from Wisconsin and Massachusetts did not indicate grouping by state. BlShV was detected in cranberry pollen, and seed transmission of up to 91% was observed. Artificial inoculation of cranberry flowers by pollination did not cause virus transmission. In some Nicotiana spp., rub inoculation of leaves with homogenized BlShV-positive cranberry flowers resulted in systemic infection. Cranberry plants recovered from symptoms the year after berry scarring occurred but continued to test positive for BlShV. The virus caused significant reduction in the average number of marketable fruit and average berry weight in symptomatic cranberry plants but recovered plants yielded comparably with healthy plants. Although recovery may limit the immediate economic consequences of BlShV, long-term implications of single- or mixed-virus infection in cranberry is unknown.

English

Cucumber mosaic virus (CMV) is an important pathogen in agricultural crops which is spread throughout the world with a wide host range. In Indonesia, CMV is also an important pathogen that infect various high economic value crops. Karanganyar regency is a horticultural production centers in Central Java. Its foremost products are cucurbits incuding cucumber (Cucumis sativus), melon (Cucumis melo), watermelon (Citrullus lanatus), pumpkin (Cucurbita moschata), chayote (Sechium edule), angled luffa (Luffa acutangula) and bitter melon (Momordica charantia). Recently, cucurbits crops in the area suffered from disease with symptoms of virus infection, especially CMV, that is, mosaic, vein clearing, vein banding, malformation, etc. The disease is detrimental to farmers. This research aimed to determine whether the causal agent of disease in the crops is CMV. For that, the authors conducted a survey in the field, picked up sample crops showing symptoms, brought the sample crops to the laboratory, and checked for the presence of CMV by triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The results showed that of the 50 sample crops including 7 species of cucurbits showing symptoms of virus infection, 12 were detected to be infected by CMV. CMV was distributed evenly in cucurbits in Karanganyar regency. Key words: Cucumber mosaic virus (CMV), cucurbits, triple antibody sandwich enzyme-linked immunosorbent assay (TAS ELISA), Karanganyar, Indonesia.

Potato virus Y strains infecting potatoes in the Msinga district in the province of KwaZulu-Natal, South Africa

Accurate identification of plant pathogens is crucial towards developing sustainable control strategies to ensure sustainable economic agricultural production. The aim of this study was to detect and characterize Potato virus Y (PVY) isolates infecting potato (Solanum tuberosum L.) in the Msinga district in the Province of KwaZulu-Natal, South Africa. Potato leaf samples exhibiting virus-like symptoms were collected from four different areas in the district. Initial detection of PVY in the leaf samples was done using triple antibody sandwich ELISA. PVY-positive samples were further tested using antibodies specific to PVY serotypes O and N. Nicotiana tabacum cv Samsun plants were individually mechanically inoculated with all 32 PVY-ELISA positive samples. Symptoms on inoculated tobacco plants were monitored over a 4-week period. They consisted of vein clearing, faint mosaic patterns, and the veinal necrosis, symptoms characteristic of PVYN, PVYNWilga and PVYNTN strains. Reverse transcription-polymerase chain reaction, using primers specific to the coat protein gene of PVY, was performed as a confirmation test on total RNA of four randomly selected PVY-ELISA positive samples, each sample representing each of the four areas surveyed. Strains PVYN and PVYO were identified. The second part of the study aimed to analyse the full genome sequences of the PVY isolates A4, KD2, MOD1 and SneP3, in order to understand the evolution of the virus in Msinga. To achieve this, total RNA, extracted from tobacco leaves (N. tabacum cv Samsun) that had been inoculated with the selected four PVY isolates, was used as a template for next generation sequencing (NGS). NGS was run on Illumina HiSeq using paired-end chemistry 125 × 125bp reads. de novo assembly of the generated reads was performed. The resulting contigs were subjected to BLAST on the GenBank database in order to identify PVY genomes. The PVY isolates were aligned with closely related non-recombinant PVY sequences comprising of the following strains: PVYN, PVYO, PVYNTN, and PVYC. Recombination events were assessed using RDP4 software. Phylogenetic results revealed that PVY isolate SneP3 belonged to the PVYNTN strain while isolate PVYMOD1 to the PVY N Wilga strain group. Recombination analyses confirmed the occurrence of PVY recombinant strains in the Msinga district. The widespread presence of PVY and occurrence of recombinant strains in Msinga has serious implications on the management of PVY diseases by small-scale farmers growing potato for a livelihood.

Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays

The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.

Diversity of Rice yellow mottle virus in Benin and Togo and Screening for Resistant Accessions

AbstractSurveys were conducted in rice fields in Benin, Cote d'Ivoire, Mali, Nigeria, Togo and Niger to assess the importance of Rice yellow mottle virus (RYMV). Diseased leaf samples were collected. In Togo, surveys were made mainly in the southern part of the country, and in Benin, all areas where rice is grown were covered. Leaf samples were serologically confirmed by ACP ELISA as bearing RYMV, propagated and all conserved. One hundred and forty‐eight (148) Beninese and 27 Togolese strains were serotyped. They were also phenotyped on three susceptible accessions including IR 64, four resistant lines with known alleles on RYMV 1 gene namely TOG 5681 (rymv 1‐3), TOG 5672 (rymv 1‐4 and RYMV 2), TOG 5674 (rymv 1.5) and Gigante (rymv 1‐2). RYMV spots with 9–100% incidences were identified. Serotyping by triple antibody sandwich (TAS) ELISA indicated that two main groups S1 and S2 coexisted in Benin with S1 being prevalent. In Togo, 26 strains were S1 and only one was S2. Phenotyping of the 148 Beninese and 27 Togolese indicated that they all attacked the three susceptible accessions, while TOG 5681 (rymv 1‐3), TOG 5672 (rymv 1‐4 and RYMV 2), TOG 5674 (rymv 1‐5) and Gigante (rymv 1‐2) remained symptomless. The use of the genes/alleles above in these countries against the disease is discussed. Three representative Beninese strains were selected to screen 48 accessions for disease resistance. Nine accessions were as susceptible as IR 64 to all strains while six (NERICA 9, NERICA 12, NERICA 13, TOG 7291, WAB56‐50, CG 14 and Moroberekan) were very resistant. Susceptibility of the six ARICA and resistance of some NERICA lines could be explained by the fact that one or both parental lines were, respectively, susceptible or resistant to the strains.

English

In order to evaluate the systemicity of BBTV from one plant of the mat to the physically attached shoots, 60 mats both of “Yangambi Km5”, Musa AAA and those of the false horn plantain “Libanga Likale”, Musa AAB showing severity levels from 0 to 5 were selected in backyards in Kisangani. In addition, 30 sucker corms per genotype were put under macro-propagation and leaf samples of lateral shoots that had emerged were tested using triple antibody sandwich-enzyme linked immuno sorbent assay (TAS-ELISA). In the backyards, for mats with no visible banana bunchy top disease (BBTD) symptoms, none of the analyzed mats with a total of 29 plants of “Yangambi Km5” and of 35 plants of “Libanga Likale” tested ELISA positive, indicating the absence of the BBTV infection. However, for the severity levels of one to five, 32 to 63.5% of plants in the mats were ELISA positive for “Yangambi Km5”, while 34.9 to 73.2% of plants from “Libanga Likale” tested positive for BBTV. After macro-propagation, 100% of lateral shoots of both cultivars at BBTD severity levels 4 and 5 tested positive. On the other hand, none of the lateral shoots at level 0 tested ELISA positive. However, for levels 1 to 3 some ELISA negative plantlets (40 to 23% for “Yangambi Km5” and 53 to 15% for “Libanga Likale”) were observed. This study indicates the need for the complete destruction of all mats harbouring plants with BBTD severity levels of 3, 4 and 5. Macro-propagation of suckers with severity level 1 symptoms could produce virus-free plantlets but ELISA testing of the lateral shoots is essential to pinpoint the virus-free plantlets. Key words: Banana bunchy top viral infection (BBTV), macro-propagation, mat, systemicity, triple antibody sandwich-enzyme linked immuno sorbent assay (TAS-ELISA).

Brachypodium distachyon is a suitable host plant for study of Barley yellow dwarf virus.

Barley yellow dwarf viruses (BYDVs) belong to the family Luteoviridae and cause disease in cereals. Because of the large and complex genome of cereal plants, it is difficult to study host-virus interactions. In order to establish a model host system for the studies on BYDVs, we examined the susceptibility of a monocot model plant, Brachypodium distachyon, to BYDV-GAV infection. Fourteen days after BYDV-GAV inoculation by aphid transmission, B. distachyon plants (inbred line Bd21-3) showed conspicuous disease symptoms such as leaf reddening, dwarfness and root stunting. Virus accumulation was detected in both shoots and roots using reverse transcription PCR and triple antibody sandwich ELISA. Compared with infected wheat plants, B. distachyon plants developed more severe disease symptoms and accumulated a higher level of BYDV-GAV. Under transmission electron microscope, we observed that virus particles accumulated in companion cells and BYDV-GAV infection was associated with the deformation of chloroplasts in the infected leaves of B. distachyon plants. Our results suggest that B. distachyon is a suitable and promising experimental model plant for the host-BYDV-GAV pathosystem and possibly for other BYDVs.

Current situation of tomato yellow leaf curl disease (TYLCD) in Antalya, Turkey

Gul-Seker M., Ekinci H., Ozturk C., Elibuyuk I.O. (2015): Current situation of tomato yellow leaf curl disease (TYLCD) in Antalya, Turkey. Plant Protect. Sci., 51: 208–213. We studied the distribution of virus/viruses causing tomato yellow leaf curl disease (TYLCD) in Antalya and to charac terise the partial genome sequences of the selected isolates. Tomato yellow leaf curl virus (TYLCV) was only detected by the triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and polymerase chain reaction (PCR). 24 and 80 greenhouses were found to be TYLCV-infected in the spring and autumn cultivations in 2011, respectively. The rate of TYLCV infection was found to be 29% in the spring cultivation and 43.7% in the autumn cultivation. The partial nucleotide sequences of the isolates were also determined.

Screening of pepper (Capsicum annuum L.) lines for resistance to an isolate of Potato virus Y (PVY) occurring in KwaZulu-Natal (KZN), Republic of South Africa

Potato virus Y (PVY) is an economically important virus of field grown solanaceous crops throughout the world. In the province of KwaZulu-Natal (KZN), Republic of South Africa (RSA), the incidence of PVY infection has been reported to significantly reduce the yield and quality of cultivated peppers. This study aims to evaluate commercially grown pepper lines for resistance against PVY. Six commercial F1 pepper lines were challenged with the PVY isolate JVW-186 using mechanical inoculation. Each line was assessed for resistance to PVY by visual screening for disease severity and quantitative triple antibody sandwich enzyme linked immunosorbent assay (TAS-ELISA) for virus load. The pepper lines were further characterized using tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR) to identify and differentiate homozygous/heterozygous resistance alleles at the pvr2 locus that confers PVY resistance. Our results identified two resistant lines (Double Up and Cecelia) and varying levels of susceptibility in the other lines challenged with PVY. The pvr2+ allele was positively identified in all the susceptible pepper lines which confirmed that the presence of this allele is dominant for PVY susceptibility. The information generated in this study can be used in breeding programs to develop PVY resistant pepper.

First Report of Grapevine leafroll-associated virus-2 and -3 in Texas Vineyards.

Grapevine leafroll disease (GLD), caused by the grapevine leafroll-associated viruses (GLRaVs, family Closteroviridae) is an important disease in all grapevine-growing regions of the world (2). It negatively affects vine vigor, fruit yield, and grape quality (e.g., sugar accumulation) (3). Typical disease symptoms include downward rolling of grape leaves accompanied by interveinal reddening in red-fruited varieties and interveinal chlorosis in white-fruited varieties (2). The state of Texas currently has over 275 commercial vineyards and acreage under grape production is expanding. Currently, there is limited information on the presence of either GLRaV-2 (genus Closterovirus) or GLRaV-3 (genus Ampelovirus) in this state. During the 2012 season, 19 individual, symptomatic grapevines (13 cv. Lenoir and 6 cv. Blanc du Bois) were sampled (14 petioles per vine) from one vineyard site in Richards, TX. Total nucleic acid was extracted from the samples as described before (5) and tested by RT-PCR using species specific primers to amplify a 334-bp fragment of the HSP70h gene of GLRaV-2 (L2 F: 5'-ATAATTCGGCGTACATCCCCACTT-3' and U2 R: 5'-GCCCTCCGCGCAACTAATGACAG-3') (1) and a 541-bp fragment of the HSP70h gene of GLRaV-3 (LC1 F: 5'-CGCTAGGGCTGTGGAAGTATT-3' and LC2 R: 5'-GTTGTCCCGGGTACCAGATAT-3') (4). Samples were also subjected to triple (TAS) and double (DAS) antibody sandwich ELISA for GLRaV-2 and GLRaV-3 using commercially available antibody test kits (AC Diagnostics, Fayetteville, AR). Five samples tested positive for GLRaV-2 and one for GLRaV-3, all from the variety Lenoir with no incidences of mixed infection. In addition to the RT-PCR and ELISA, the presence of GLRaV-2 and GLRaV-3 was confirmed by direct sequencing of select RT-PCR products, which was purified using the QIAquick PCR Purification kit (Qiagen Inc., CA). The sequencing took place at the Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA. GLRaV-2 isolate TX12 (GenBank Accession No. KF417612) and GLRaV-3 isolate TX9 (KJ545571) shared 85 to 100% and 94 to 100% nucleotide identity and 74 to 100% and 82 to 100% amino acid identity, respectively, with previously reported isolates from around the world. All samples tested negative for GLRaV-1, -4, -4 strain 5, and -4 strain 9 (4), suggesting that some of the symptomatic vines may have a different disease or abiotic disorder, such as a nutrient deficiency. To our knowledge, this is the first report of GLRaV-2 and GLRaV-3 in the state of Texas.

English

Banana bunchy top disease (BBTD) was first identified in DR Congo in 1958. Previously, the disease’s distribution throughout the Congo basin had not been studied, so an initial study, to determine the incidence and severity of BBTD in banana and plantain producing regions of Oriental province, was carried out during 2009 to 2010. Three hundred (300) farms were surveyed across 4 districts and 19 territories, with 30 mats assessed per farm. Visible disease symptoms were recorded and serological tests using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) were carried out on collected samples. Additional surveys were conducted during 2010 to 2012 in Maniema, Northern Katanga, Eastern and Western Kasai, Bandundu and Equateur provinces to assess the distribution of the aphid vector (Pentalonia nigronervosa), BBTD incidence and severity. 92% of mats observed across Oriental province manifested BBTD symptoms but severity levels were low. All plantain and banana cultivars grown in farmers’ fields were susceptible to the disease. The vector, P. nigronervosa, was found on 89% of all assessed mats. In Tshopo district, all samples collected on plants showing disease severity scores 2, 4 and 5 tested positive for the presence of the virus. However, only 48% of plants with severity score 1 and 33% of plants with score 3 gave positive TAS-ELISA results. More importantly, 40% of symptomless plants (score 0) tested positive. The average BBTD incidences in Bandundu, Equateur, Eastern and Western Kasai, Katanga and Maniema were lower than levels observed in Oriental province. The lowest incidence levels were observed in Equateur (43%) and Katanga (35%). Although, BBTD is widespread in the surveyed provinces, the generally observed low severity levels result in limited impact on production. The generalized spread of BBTD in surveyed areas nevertheless underlines an urgent need to identify virus-free plants for multiplication and distribution of disease-free materials to small-scale farmers.

Pentalonia nigronervosaCoquerel andPentalonia caladiivan der Goot (Hemiptera: Aphididae) and Their Relationship to Banana Bunchy Top Virus in Micronesia

Geographical distribution of Pentalonia nigronervosa and P. caladii, and incidence of banana bunchy top virus (BBTV) were determined in Micronesia. Aphids and plant tissues were collected from banana and nonbanana hosts of Petrtalonia aphids in Palau, Yap, Guam, Rota, Tinian, Saipan, Pohnpei, Kosrae, and Majuro. Aphids were identified based on host species from which they were collected and on morphometric analysis of length of ultimate rostral segment. All plant samples were tested for presence or absence of BBTV using a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). TAS-ELISA analysis confirmed BBTV to be present on Guam, Saipan, and Rota, but BBTV was not detected elsewhere in Micronesia.

The status of Cucumber vein yellowing virus in Iran

Yellowing symptoms of greenhouse- and open field-grown cucurbit crops are becoming increasingly important in many cucurbit growing regions of the world, and particularly in Iran. A survey was conducted from 2011 to 2012 in eight major cucurbit growing regions in Iran. Yellowing and specifically vein clearing symptoms were observed in many cucumber plants grown in greenhouses and open fields, suggesting the presence of Cucumber vein yellowing virus (CVYV, genus Ipomovirus , family Potyviridae ). The identification of CVYV was carried out with a specific triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and reverse transcription (RT)-PCR. CVYV was detected in 42% of the collected samples, in all surveyed provinces, except Bushehr. CVYV was also detected in melon and cucumber crops grown in open fields. These results indicate that CVYV is widely distributed on these two cucurbit species in the major cucumber growing areas of Iran. CVYV positive samples were also tested, using DAS-ELISA, for the presence of Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV), two criniviruses reported previously to occur in Iran. Double or triple infections of CCYV and CYSDV occurred in 49 of 166 of the CVYV-infected plants. The CVYV and CCYV combined infections were more prevalent than CVYV and CYSDV combined infections. TAS-ELISA positive samples were used to mechanically inoculate healthy cucumber plants, and mild vein yellowing was observed on the inoculated leaves. Identical symptoms were also observed on whitefly inoculated healthy cucumber plants. The presence of CVYV in mechanically and whitefly inoculated plants was confirmed by TAS-ELISA and RT-PCR. Sequence analysis revealed that the Iranian isolate of CVYV was more closely related to Spanish isolates than to isolates from Jordan. Phylogenetic analysis showed that CVYV isolates can be divided into two phylogenetic groups (I and II). Despite the close vicinity of Jordan to Iran, the Iranian CVYV isolate clustered with Spanish isolates in group I and not with the Jordanian isolates of group II.

First Report of Capsicum chlorosis virus Infecting Waxflower (Hoya calycina Schlecter) in the United States.

In February 2013, an ornamental waxflower (Hoya calycina Schlecter) with leaves displaying concentric chlorotic and necrotic rings surrounding sunken, necrotic lesions typical of tospovirus infection was observed at a community garden in Honolulu, HI. Symptomatic leaf tissue tested negative for Tomato spotted wilt virus (TSWV), a common tospovirus in Hawaii, using a TSWV ImmunoStrips (AgDia, Elkhart, IN) assay following the manufacturer's instructions. Double-stranded RNAs were isolated from a symptomatic leaf and reverse transcribed using random primers (2). The cDNA was then used as template in a universal tospovirus PCR assay using primers gL3637 and gL4435c, which amplify sequences of the L segment encoding the RNA-dependent RNA polymerase of tospoviruses (1). An ~800-bp product was amplified and cloned using pGEM-T Easy (Promega, Madison, WI). Three clones were selected and found to be identical by dye-terminator sequencing performed at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. Following primer sequence trimming, the 773-bp sequence (GenBank Accession No. KF030938) was found to be 97, 88, and 87% identical to Capsicum chlorosis virus (CaCV; a tentative species in the family Bunyaviridae, genus Tospovirus) strains Ch-Har (GU199334), TwTom1 (HM021140), and AIT (DQ256124), respectively. To confirm the presence of CaCV, the cDNA was also used as template in a universal tospovirus PCR assay with primers 3'T12 and TsMCR2 which amplify a region of the S segment of tospoviruses (3). The amplification product from this assay was cloned and sequenced as described above and found to be 93 to 98% identical to CaCV nucleotide sequences present in GenBank. Attempts to detect the CaCV strain in waxflower using a watermelon silver mottle virus and groundnut bud necrosis virus triple antibody sandwich ELISA (AgDia) were unsuccessful. No other plants in the community garden had typical tospovirus-like symptoms; however, samples from tomato (Solanum lycopersicum L.; two samples), chili pepper (Capsicum spp.; four samples), eggplant (Solanum melongena L.; one sample), and passionfruit (Passiflora edulis Sims; one sample) with virus-like symptoms were collected from the garden and had RNA isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). No tospoviruses were detected in any of these samples with the RT-PCR assay using primers gL3637 and gL4435. The waxflower plant infected with CaCV was immediately removed by community garden members and destroyed, preventing any additional serological or biological assays to be performed. CaCV is transmitted by several species of thrips, including Thrips palmi, which is present in Hawaii. Waxflower is not native to Hawaii and it is unclear whether CaCV entered Hawaii in this plant or whether it was infected by viruliferous thrips. A survey for CaCV in known hosts is essential to determine the geographic distribution of CaCV in Hawaii, as this virus poses a considerable threat to tomato, chili pepper, and phalaenopsis orchid production in Hawaii and the United States.

Monoclonal antibody-based serological methods for maize chlorotic mottle virus detection in China

Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327680, 1:64000, and 1:3276800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China.

Detection and partial molecular characterization of atypical plum pox virus isolates from naturally infected sour cherry

Atypical isolates of plum pox virus (PPV) were discovered in naturally infected sour cherry in urban ornamental plantings in Moscow, Russia. The isolates were detected by polyclonal double antibody sandwich ELISA and RT-PCR using universal primers specific for the 3'-non-coding and coat protein (CP) regions of the genome but failed to be recognized by triple antibody sandwich ELISA with the universal monoclonal antibody 5B and by RT-PCR using primers specific to for PPV strains D, M, C and W. Sequence analysis of the CP genes of nine isolates revealed 99.2-100 % within-group identity and 62-85 % identity to conventional PPV strains. Phylogenetic analysis showed that the atypical isolates represent a group that is distinct from the known PPV strains. Alignment of the N-terminal amino acid sequences of CP demonstrated their close similarity to those of a new tentative PPV strain, CR.

Occurrence and Genetic Diversity of Winona-Like Plum pox virus Isolates in Russia.

In studying the distribution and genetic diversity of Plum pox virus (PPV) in Russia, over a dozen new PPV isolates belonging to the strain Winona (PPV-W) were identified by immunocapture reverse-transcription polymerase chain reaction with the PPV-W-specific primers 3174-SP-F3/3174-SP-R1. Isolates were detected in two geographically distant regions of European Russia (Northern Caucasus and Moscow regions) in naturally infected plum (Prunus domestica), blackthorn (P. spinosa), Canadian plum (P. nigra), and downy cherry (P. tomentosa). The new PPV-W isolates were shown to be serologically related but not identical by triple-antibody sandwich enzyme-linked immunosorbent assay and Western blotting analysis using the monoclonal antibody (MAb) 5B-IVIA and MAbs specific to the N-terminal epitopes of PPV-W isolate 3174. Analysis of nucleotide and deduced amino acid sequences of the (C-ter)NIb-(N-ter)CP genome region indicate great genetic diversity among isolates, with phylogenetic analysis revealing seven clades. Isolates P1 and P3 found in plum in the south of Russia clustered closely with the putative ancestral PPV-W isolate LV-145bt from Latvia, while isolate 1410-7 found in P. nigra in Moscow appears to be closely related to the Canadian isolate W3174. The data obtained indicate wide dissemination of PPV-W isolate in stone fruit in the European part of the former USSR.

Three Distinct Nanoviruses, One of Which Represents a New Species, Infect Faba Bean in Ethiopia.

In all, 70 of 296 leaf samples (23.6%) collected from faba bean (Vicia faba) plants showing leaf yellowing and stunting in Ethiopia gave nanovirus-positive reactions when studied by triple-antibody sandwich enzyme-linked immunosorbent assay using broad-spectrum monoclonal antibodies (MAbs) specific to nanoviruses. Further analysis of these samples with seven discriminating MAbs revealed contrasting epitope profiles that were categorized into roughly three serogroups, designated A, B, and C. Serogroup A was found in 89% of the nanovirus-positive samples whereas serogroups B and C were infrequently encountered. Sequence analysis of DNA-S and DNA-U1 of serogroup A, B, and C isolates suggested that each represents a distinct nanovirus species. Serogroup A comprised isolates of Faba bean necrotic stunt virus reported earlier only from Ethiopia and Morocco. The DNA-R, -S, -U1, and -U2 sequences of a serogroup B isolate closely resembled those of Faba bean necrotic yellows virus, providing first molecular evidence for its occurrence in Ethiopia. Sequence analysis of the eight genomic DNAs of a representative serogroup C isolate (Eth-231) showed that it shared overall nucleotide and amino acid sequence identities of only ≤70 and ≤74%, respectively, with other nanoviruses. This suggests that Eth-231 represents a new nanovirus species, for which the name faba bean yellow leaf virus is proposed.

Monoclonal Antibodies Against the Whitefly-Transmitted Tomato Yellow Leaf Curl Virus and Their Application in Virus Detection

Tomato yellow leaf curl virus (TYLCV) is a species of the family Geminiviridae, causing serious yield losses in tomato production. The coat protein (CP) gene of TYLCV isolate SH2 was expressed in Escherichia coli BL21 (DE3) using pET-32a as the expression vector. The recombinant protein was purified through Ni+-NTA affinity column and used to immunize BALB/c mice. Three hybridoma cell lines (2B2, 2E3 and 3E10) secreting monoclonal antibodies (MAbs) against TYLCV CP were obtained by fusing mouse myeloma cells (Sp 2/0) with spleen cells from the immunized BALB/c mouse. The titers of ascitic fluids of three MAbs ranged from 10−6 to 10−7 in indirect-ELISA. Isotypes and subclasses of all the MAbs belonged to IgG1, k light chain. Triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) showed that the MAb 3E10 could react with five begomoviruses infecting tomato, while the other two (2B2 and 2E3) mainly reacted with TYLCV. TAS-ELISA was set up using the MAb 3E10, and the established method could successfully detect virus in plant sap at 1:2 560 (w/v, g mL−1). Detection of field samples showed that begomoviruses were common in tomato crops in Zhejiang Province, China.