Direct Antigen-coated Enzyme-linked Immunosorbent Assay Research Articles

First report of Tobacco streak virus infecting Lablab purpureus

Lablab purpureus (lablab bean) is a highly drought-tolerant legume crop widely cultivated throughout the tropics. Leaf veinal necrosis and necrotic spotting and necrotic streaks on stems and petioles were observed on L. purpureus in farmers' fields in the Chittore district, Andhra Pradesh, India in December 2012 (Fig. 1). Based on the symptomatology, infection by Tobacco streak virus (TSV) was suspected (Bhat et al., 2). The infected leaves tested positive by direct antigen-coated enzyme-linked immunosorbent assay (Bhat et al., 1) using TSV polyclonal antibodies (provided by P. Lava Kumar, IITA, Ibadan, Nigeria). Total RNA was isolated from infected leaves using the TRIzol® reagent method and RT-PCR amplification was done using a novel pair of primers (CP-F-AGCAGATGCCCAACTTGTTT, CP-R-AAGGGAGCTGGTTTGGATA) designed to amplify 604 bp product from the coat protein gene of TSV. The primers amplified a product of the expected size from symptom-bearing but not healthy plants (Fig. 2). The PCR product was cloned, sequenced and deposited in GenBank (Accession No. KC683811). The Indian isolate shared 98.5-99.3% nucleotide identity with other TSV isolates (AY940151, HQ324116 and HQ324117) To the best of our knowledge this is the first report of the natural occurrence of TSV on L. purpureus. Wilting and necrosis of plants leading to a reduction in yield was observed in L. purpureus fields naturally infected with TSV. TSV spreads easily in fields through pollen assisted by thrips and by mechanical transmission. Intercropping of L. purpureus with groundnut, cowpea and other susceptible vegetable crops is common in India. This increases the possibility TSV being introduced into L. purpureus crops and transmission between crops. Implementing control measures for TSV is difficult due to the wide host range of the virus and the presence of efficient vectors throughout the crop period (Prasada Rao et al., 3).

Transgenic chickpea expressing a recombinant human α1-proteinase inhibitor (α1-PI) driven by a seed-specific promoters from the common bean Phaseolus vulgaris (L.)

The 5′ regulatory sequences of seed-storage protein genes, arcelin 5-I (Arc), phaseolin (Phas) of common bean (Phaseolus vulgaris L.) and legumin (Leg) gene of Cicer arietinum (L.) were evaluated for seed-specific expression of β-glucuronidase (uidA) and recombinant human α1-proteinase inhibitor (α1-PI) in transgenic chickpea. Histochemical assay of transformed plants developed with seed-specific promoters, showed GUS expression in seeds and not in other plant tissues. Fluorometric assay of β-glucuronidase revealed that phaseolin promoter with arcelin 5′ UTR (pPAG) resulted into maximum GUS activity in seeds, followed by somatic tissues and minimal expression in leaves. The expression profile of GUS driven by different seed-specific promoters in chickpea, are in order phaseolin > arcelin > legumin respectively. RT-PCR analysis confirmed that transcripts of β-glucuronidase and α1-PI genes, driven by the phaseolin promoter are stable in chickpea and their accumulation is confined into the seed tissues. Analysis of α1-PI expression in seeds of transgenic chickpea was performed by direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA), residual porcine pancreatic elastase activity assay and Western immunoblotting. Results of DAC-ELISA showed that modified α1-PI gene driven by the phaseolin promoter with arcelin 5′ UTR showed maximum accumulation of α1-PI up to 1.95 μg mg−1 of fresh weight in seeds. Biological activity of recombinant α1-PI, confirmed by elastase inhibition assays exhibiting 0.264 μg mg−1 of active protein. Western immunoblot analysis confirmed the molecular mass of plant-expressed recombinant α1-PI of molecular mass ~50 kDa in the seeds of transgenic chickpea.

Immunodiagnostics of cucumber mosaic virus using antisera developed against recombinant coat protein

Cucumber mosaic virus (CMV), causing mosaic disease in banana cultivars, is the most widespread virus after Banana bunchy top virus. The entire coat protein (CP) gene of CMV was amplified by polymerase chain reaction (PCR) and engineered to be expressed in bacterial expression vector pQE30, under the control of isopropyl-thio-β-d galactopyranoside (IPTG)-inducible T7lac promoter. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed expressed CP as ∼25 kDa protein. Expression conditions for maximum recovery of soluble recombinant protein were standardised. The recombinant CP (r-CP) fused with His-tag was purified from Escherichia coli using nickel-nitrilotriacetic acid (Ni-NTA) resin and used as an antigen for the production of polyclonal antisera. This antiserum was used to develop enzyme-linked immunosorbent assay (ELISA) and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection of CMV isolates. The antiserum had a titre of 1:8000 when tested with r-CP. The reaction of polyclonal antisera was much better than that of commercial antibody. On comparing IC-RT-PCR with direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA) using polyclonal antisera, IC-RT-PCR was found to be more sensitive, hence can be used to detect the presence of virus in tissue culture based banana plant as well as in various host plants.

Field performance of cowpea genotypes grown under virus pressure in Puerto Rico

Cowpea [Vigna unguiculata (L.) Walp] is an important grain legume in many regions of the tropics. However, viral diseases, particularly Cucumber mosaic virus (CMV) and Blackeye cowpea mosaic virus (BlCMV), can be a limiting factor in cowpea production. We evaluated under virus pressure the performance of four PI’s (441917, 441919, 612607, 180014) and a commercial cultivar (Coronet), in Puerto Rico during 2007-2008. Viral infection of plants with BlCMV and CMV in these experiments occurred naturally through insect vectors from mechanically inoculated spreader-row plants, as determined by direct antigen-coated enzyme-linked immunosorbent assay (DACELISA). PI441917 had the highest grain yield (3682.8 kg ha-1) among all genotypes except for the virus resistant control PI 612607, which was not significantly different. PI’s 180014 and 612607 had the highest seed protein concentration. The cultivar Coronet attained midbloom and maturity earlier than the other genotypes. These results confirm previous studies which showed that PI 441917 is an excellent yielder when plants are grown under virus pressure. This accession should be useful in cowpea breeding programmes to help control yield loses by CMV and BlCMV.Key Words: Blackeye cowpea mosaic virus, Vigna unguiculata

Serological Detection of Cymbidium mosaic and Odontoglossum ringspot viruses in Orchids with Polyclonal Antibodies Produced Against Their Recombinant Coat Proteins

AbstractCymbidium mosaic and Odontoglossum ringspot viruses infecting orchids were identified by coat protein (CP) properties. The Cymbidium mosaic virus (CymMV) CP gene is 672 nt long, potentially encoding 223 amino acids (aa). The Odontoglossum ringspot virus (ORSV) CP gene is 477 nt long, potentially encoding 158 aa. The CP gene of CymMV and ORSV isolates originating from different locations was highly conserved both at the nucleotide and amino acid levels (94–100%). Polyclonal antibodies against CymMV and ORSV were separately produced using bacterially expressed recombinant CP as immunogens. Antisera to CymMV (titre 1 : 2000) and ORSV (titre 1 : 250) detected the viruses by direct antigen‐coated enzyme‐linked immunosorbent assay (DAC‐ELISA) in orchid samples collected from Sikkim, India. Survey results indicated the prevalence of mixed infection of CymMV and ORSV in Cymbidium spp. The immunoreagents we developed will be useful for virus indexing in orchid certification programmes.

Transmission of Cucumber Mosaic Virus (CMV) infecting banana by aphid and mechanical methods

Banana is the premier fruit of the Asia and pacific region. It is the fourth most important food crop after rice, wheat and maize. In Tamil Nadu, banana is grown in 92,200 hectares with an annual production of 48.56 million tones. Among all biotic constraints, viruses cause huge economic loss to the crop. Some of the major diseases are caused by viruses which include Banana Bunchy Top Virus (BBTV), Cucumber Mosaic Virus (CMV), Banana Streak Virus (BSV), Banana Bract Mosaic Virus (BBMV) have been reported in our country. CMV virus concentration in host (Musa) tissue is very less. So it is necessary to transfer this virus to good propagation host such as Nicotiana sp for purification. The purpose of the study was the transmission of CMV infecting banana into propagation host either by aphid or mechanically. The transmission was confirmed through Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA).

Production of Polyclonal Antibodies Using Recombinant Coat Protein of Papaya ringspot virus and their Use in Immunodiagnosis

Production of polyclonal antibodies requires large amount of purified virus that can be avoided by the use of recombinant coat protein (CP). Recombinant CP of Papaya ringspot virus (PRSV) was thus used for the production of polyclonal antibodies as the virus purification from papaya tissues provides low virus yields. CP was expressed as a fusion protein (∼72 kD) containing a fragment of E. coli maltose binding protein. Polyclonal antibodies from rabbits immunized with the fusion protein, successfully detected natural infection of PRSV in papaya and cucurbits samples collected from different locations at 1:4000 dilution in direct antigen-coated enzyme-linked immunosorbent assay.

Elimination of mixed infection of Cucumber mosaic and Tomato aspermy virus from Chrysanthemum morifolium Ramat. cv. Pooja by shoot meristem culture

Mixed infection of Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) was detected in Chrysanthemum morifolium Ramat. cv. Pooja plants by direct antigen coating enzyme-linked immunosorbent assay (DAC-ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Elimination of CMV and TAV was achieved by culturing 0.3 mm long shoot meristem of infected plants on MS medium supplemented with 3.0 mg/l BAP and 0.5 mg/l NAA. The regenerated plants were indexed by DAC-ELISA and confirmed by RT-PCR. A total of 78.1% CMV and TAV-free shootlets were obtained from the regenerated shoot meristem as indexed by DAC-ELISA, of which only 65.6% were found truly virus-free when confirmed by RT-PCR. Virus-free shootlets were rooted on half MS medium and acclimatized under glasshouse. These plants showed better growth and quality of blooms as compared to diseased ones.

Occurrence of a new necrosis viral disease of blackgram (Vigna mungo) and identification using electron microscopy and ELISA technique

A new necrosis viral disease was observed in blackgram, showed brown necrotic rings along with veinal and stem necrosis. The virus was mechanically inoculated on the local lesion host, cowpea cv. 152 and maintained in the local lesion host throughout the study. Yield studies under pot culture experiment showed 10- to 30-day-old plants were highly susceptible and the yield became almost nil. By using Transmission Electron Microscope (TEM) and indirect Direct Antigen Coated-Enzyme Linked Immuno-Sorbent Assay (DAC-ELISA) studies the virus was identified as Tobacco streak virus (TSV). The ultraviolet absorbance of the purified virus was measured and the ratio of A260/A280 was determined as 1.41. Polyclonal antiserum was raised against blackgram necrosis virus in New Zealand white rabbit and the titre value was determined as 1: 200. Direct antigen coating-ELISA was used to detect the virus concentration in various plant parts and stem portion recorded maximum virus concentration. TSV in blackgram was not transmitted through seeds.

Note: Comparison of antibody- and genome-based diagnostic techniques forSugarcane mosaic virus in sugarcane

Different antibody-based diagnostic techniques such as direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA), electroblot immunoassay, immunosorbent electron microscopy and dot blot immunoassay were compared with a genome-based technique,viz., reverse transcriptase polymerase chain reaction (RT-PCR) againstSugarcane mosaic virus (SCMV) in different sugarcane cultivars expressing predominant and less predominant symptoms as well as in asymptomatic ones. Polyclonal antiserum raised against SCMV antigen purified from sugarcane cv. CoC 671 was used. All of the antibody-based tests reacted positively with plants showing predominant foliar symptoms; however, the sensitivity was not uniform, with samples from the plants showing less predominant symptoms and in asymptomatic plants. The RT-PCR assay method was more sensitive in detecting the virus from plants of less predominant symptoms also. Hence, a combined diagnostic system utilizing antibody-based techniques can be employed for mass screening and in quarantine operations against the virus, but in doubtful cases RT-PCR can be employed. Among the different sugarcane tissues, SCMV was diagnosed in the leaf lamina, bud, leaf sheath and midrib tissues, but root and pith tissues were negative to the virus in RT-PCR assay. In RT-PCR, 870 bp size cDNA was amplified from infected sugarcane with SCMV specific primer.

Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India.

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV-AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV-AP. The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV. In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

First Report of Peanut stunt virus in Perennial Peanut in North Florida and Southern Georgia.

In spring 2001, symptoms similar to aphid-vectored peanut stunt disease caused by Peanut stunt virus (PSV) were observed on perennial peanut (Arachis glabrata) cv. Floragraze in Jackson and Gulf counties, FL and Lowndes County, GA. Symptoms observed in commercial hay fields and at the North Florida Research and Education Center in Marianna and Quincy included malformed leaves, plant chlorosis, leaf mottling, and stunted plants, which resulted in reduced foliage yield. Leaf symptoms were visible throughout the growing season. Stunting was more common in spring and early summer. No symptoms were seen on rhizomes. Diagnosis of PSV (genus Cucumovirus) from symptomatic leaves and rhizome materials of 12 plants was confirmed by a direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). ELISA tests were repeated three times. Antibodies specific to the Clemson isolate, serotype E, were obtained from Clemson University, Clemson, SC. DAC-ELISA (1) values of 0.1 A405 above the healthy control for perennial peanut were considered positive for foliage and rhizome material tested. ELISA values ranged from 0.4 to 2.1. The mean ELISA value of the positive controls was 2.65. Symptomatic plants were also tested with ELISA using available antibodies from Agdia Inc., Elkart, IN, for Tomato spotted wilt virus, from ATCC for Peanut stripe virus, and from Clemson University (Cowpea isolate) for Cucumber mosaic virus, but all results were negative. To our knowledge, this is the first report of PSV on perennial peanut in Florida and southern Georgia. At this time, it is not known what role perennial peanut may play as a reservoir of the virus in the vicinity of peanut fields. Little is known about the potential for forage production loss and stand longevity. Next season, molecular detection techniques and epidemiological studies on peanut and perennial peanut will be conducted to ascertain the incidence and possible impact of PSV in Georgia and Florida. Reference: (1) A. G. Gillaspie, Jr. et al. Plant Dis. 79:388, 1995.

Resistance to Cucumber mosaic virus in Cowpea and Implications for Control of Cowpea Stunt Disease.

Cucumber mosaic virus (CMV) and Blackeye cowpea mosaic virus (BlCMV) interact synergistically in dually infected plants of cowpea (Vigna unguiculata subsp. unguiculata) to cause cowpea stunt disease, the most damaging viral disease of this crop in the U.S. Sources of resistance to BlCMV are known and are present in cultivars of cowpea such as Pinkeye Purple Hull-BVR. However, no sources of CMV resistance have been found previously in cowpea. In 1998, PI 441918, a cowpea line growing in regeneration plots, was observed to have few viral symptoms, was not infected with BlCMV, and had a low titer of CMV when tested using direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA). In greenhouse tests, infection of PI 441918 with CMV resulted in a lower titer of virus if the inoculated plants were from white seeds of PI 441918 than if the plants were from tan seeds of this PI, and a lower titer of virus than plants of the susceptible cultivar Coronet. In the field, with CMV- and BlCMV-infected plants of Coronet in spreader rows, plants from white seed of PI 441918 had no infection with BlCMV and a low infection rate with CMV. PI 441918 offers a high level of resistance to BlCMV and moderate resistance to CMV, which are important characteristics in a parental line to develop cultivars of cowpea resistant to cowpea stunt disease.

An Immunity (QL3) Breaking Strain of Sorghum Mosaic Potyvirus in India.

Sorghum (Sorghum bicolor) line Tx2786 is immune to strains or isolates of sorghum mosaic potyvirus (SrMV strains SCH and SCH) but susceptible to strains of maize dwarf mosaic (MDMV) and sugarcane mosaic (SCMV) potyviruses (1,2). When grown in proximity to sugarcane, Tx2786 was infected with a virus that caused mosaic and necrotic symptoms during the postrainy season of 1994 (14 to 30°C) at Parbhani (Maharashtra, India). Leaf-dip electron microscopy performed on virus-infected sorghum and sugarcane tissues revealed particles, 720 × 12 nm, typical of a potyvirus. A potyvirus isolate (designated IBS) was readily transmitted mechanically from field-infected sorghum (Tx2786) and sugarcane (Saccharum officinarum cv. Co.740) to 18 glasshouse-grown sorghum inbred lines. All sorghum lines (QL-3 from India and Tx2786), including those resistant to SrMV, SCMV, MDMV, and johnsongrass mosaic potyvirus (JGMV), were infected. IBS caused necrosis in Cargill-40, Hegari, PI-35038, RTx09, Tx2786, QL-3 from India, and Redlan lines and mosaic symptoms in RTx430, SA-394, Atlas, Martin, BTx3048, Caprock, NM-31, QL-11, SCO-175-14E, Rio, and BTx623 lines. Sorghum reactions to sorghum and sugarcane virus isolates were similar. IBS also infected maize (Zea mays cv. African Tall), johnsongrass (S. halepense cv. 81, symptomless infection), and sorghum (S. bicolor cv. CS-3541) but not prosomillet (Panicum miliaceum), fingermillet (Eleusine coracana cv. IE-2540), pearl millet (Pennisetum typhoides cv. NHB-3), or oat (Avena sativa cv. Kent) when sap transmitted. IBS also was transmitted by the corn leaf aphid (Rhopalosiphum maidis) in a nonpersistent manner from sorghum and sugarcane to all 18 sorghum lines in a glasshouse. Based on direct antigen-coating enzyme-linked immunosorbent assay (DAC-ELISA), serologically specific electron microscopy, and Western blot tests, IBS was serologically related to SrMV strains SCH and SCI (2-4) but not to MDMV strains A, D, and E; SCMV strains A, B, D, SC, and MDB; or JGMV type strain from Australia and strain O from Texas (2). IBS induced formation of cytoplasmic cylindrical inclusions consisting of pinwheels associated with long, straight, laminated aggregates (subdivision type II) in sorghum and sugarcane host cells. Purified IBS contained one major polypeptide of 40 kDa and a ribonucleic acid of 3.0 × 106 Da. Polyclonal antiserum to IBS was produced in rabbits and used in DAC-ELISA to confirm the identity of IBS in sugarcane, sorghum lines, and other test plant species. On the basis of biological and serological properties, IBS isolated from field-infected sorghum grain Tx2786 and sugarcane cv. Co.740 is identified as an immunity (QL3) breaking strain of SrMV (SrMV-IBS).

RT-PCR Method for Detecting Cowpea Mottle Carmovirus in Vigna Germ Plasm.

A highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) method was developed to detect cowpea mottle carmovirus (CPMoV) in newly acquired germ plasm of Vigna spp. It detected virus in tissues diluted up to 10-9. The preferred primers were designed from the RNA replicase cDNA sequence of CPMoV. These primers were able to detect CPMoV in plants infected with 10 different isolates of the virus. There were no cross-reactions with either bean mild mosaic or melon necrotic spot carmoviruses or any of the common cowpea viral pathogens tested. The RT-PCR method was up to 105 times more sensitive than direct antigen coating enzyme-linked immunosorbent assay (DAC-ELISA) in detecting CPMoV. The RT-PCR method gave no false positive reaction as is sometimes seen with ELISA.

Antigenic Diversity Among Peanut Stripe Potyvirus (Family Potyviridae) Isolates

Abstract Several geographically and biologically distinct isolates of peanut stripe Potyvirus (PStV) were tested against available polyclonal and monoclonal antibodies. In direct antigen-coated, enzyme-linked immunosorbent assay (DAC-ELISA), the polyclonal antisera prepared against the purified virus reacted with all the isolates tested. However, a monoclonal antibody, 3AB5, also prepared against the purified virus showed dramatic difference in its reactivity to the same isolates. Tested against blotch isolates from Georgia, Virginia, and Florida, 3AB5 reacted with two of 10 isolates. The reaction was either strongly positive or negative. Antibody 3AB5 did not react with a necrotic strain of PStV from Taiwan. Results indicate the existence of antigenic diversity among PStV isolates.

First Report of Peanut Stunt Cucumovirus Naturally Infecting Desmodium sp.

Plant species in the genus Desmodium (Fabaceae) are used as forage and cover crops and include a number of common weeds such as beggarweed (D. tortuosum) and beggarlice (D. intortum). Accessions of the genus are part of the plant genetic resources collection maintained at Griffin, GA. Peanut stunt cucumovirus (PSV) was detected in naturally infected plants of Desmodium sp. PI 322505 (from Brazil) in a germ plasm regeneration plot by a direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA) with an antiserum against PSV strain ER (subgroup I) originally isolated from cowpea in Georgia. The infected plants showed mild mosaic symptoms. Indicator host studies in the greenhouse revealed symptoms characteristic of PSV on Nicotiana tabacum cv. Burley 21 (ringspots and oak leaf pattern), Chenopodium album subsp. amaranticolor (chlorotic local lesions), and Vigna unguiculata (chlorotic spots followed by systemic mild mosaic). These symptomatic indicator plants tested positive for PSV by DAC-ELISA. Greenhousegrown plants of D. incanum (kaimi-clover) and D. uncinatum (Spanish tick-clover) were inoculated with the field isolate and the plants were tested for PSV by DAC-ELISA (10 infected of 10 tested and 3 infected of 9 tested, respectively). The PSV isolate infecting Desmodium spp. was found to contain satellite RNA and it generated the predicted products in reverse transcription-polymerase chain reactions (RT-PCRs) with primers based on specific PSV-ER sequences. The RT-PCR products were confirmed by restriction-enzyme digestion (1). This is the first report of PSV naturally infecting a member of the genus Desmodium. Because some members of this genus may grow as perennial weeds near peanut, cowpea, or other host crops, this genus may serve as an alternate/overwintering host for the virus. Reference: (1) R. A. Naidu et al. Phytopathology 85:502, 1995.

Seedborne Viruses in Preintroduction Cowpea Seed Lots and Establishment of Virus-Free Accessions

Seeds from 60 cowpea preintroductions from Botswana, India, and Kenya were increased at the University of California Riverside. Second generation seed were planted in insect-free greenhouses at two locations and resulting seedlings were assayed by direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) for the presence of eight seedborne viruses. By visual selection and DAC-ELISA, 10 virus-free mother plants for each of the 60 accessions were established. The seedlots from these mother plants were subsquently planted in isolation plots at St. Croix, Virgin Islands. Seedlings from the St. Croix seed increase were observed and tested by DAC-ELISA and were found to have remained free from viruses during this field exposure. One or more of the following viruses were detected in 40 of the 60 preintroductions: 32 containing cowpea aphid-borne mosaic potyvirus (possibly blackeye cowpea mosaic potyvirus in some cases); 23 with cowpea severe mosaic comovirus; 22 with southern bean mosaic sobemovirus; seven with cucumber mosaic cucumovirus; and seven with cowpea mottle carmovirus. None were found to contain ELISA-detectable cowpea mosaic comovirus or cowpea mild mottle carlavirus. Twenty preintroductions were free of ELISA-detectable seedborne viruses. Virusfree experimental seed lots were produced for limited use by interested cowpea breeders