Article

Monoclonal antibody-based diagnostic assays for pepper mild mottle virus

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Abstract

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for pepper mild mottle virus (PMMoV) detection were developed based on monoclonal and polyclonal antibodies produced in this study. Production of anti-PMMoV monoclonal antibodies (MAbs) was achieved using the hybridoma technique and a recombinant protein of the PMMoV coat protein as the antigen. Production of a polyclonal antibody (PAb) against the recombinant coat protein of PMMoV was carried out in a New Zealand White rabbit. The PAb was specific to five tobamoviruses consisting of PMMoV, tobacco mosaic virus (TMV), odontoglossum ringspot virus (ORSV), tomato mosaic virus (ToMV) and ribgrass mosaic virus (RMV). Two TAS-ELISA protocols were developed based on the characteristics of the antibodies produced. One protocol, based on MAb TOBA1, could detect five tobamoviruses (TMV, PMMoV, ORSV, ToMV, and RMV), four of which infect peppers. In the other protocol, TOBA10 was used to specifically detect PMMoV. We also successfully developed an IC-RT-PCR procedure using TOBA1. The sensitivity of the IC-RT-PCR (1:2,000,000) was much higher than TAS-ELISA (1:5120) for detection of the virus in plant sap. We anticipate these assays to facilitate the screening process of tobamovirus-resistant pepper cultivars in breeding programs.

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... In a few initial studies, tissue blot immunobinding assay (TBIA) and dot blot immunobinding assay (DBIA) using PMMoV-specific polyclonal antibodies were used for PMMoV detection in plant tissues (87,91,92). In a recent development, monoclonal antibodies (MAbs) were developed for PMMoV detection using hybridoma technology (93). Utilizing these MAbs, the triple antibody sandwich ELISA (TAS-ELISA) procedure was developed for PMMoV detection in plant sap, with sensitivity up to 1:5120 dilution (93). ...
... In a recent development, monoclonal antibodies (MAbs) were developed for PMMoV detection using hybridoma technology (93). Utilizing these MAbs, the triple antibody sandwich ELISA (TAS-ELISA) procedure was developed for PMMoV detection in plant sap, with sensitivity up to 1:5120 dilution (93). ...
... These methods typically involve the use of primers targeting the coat protein (CP) or RNA-dependent RNA polymerase (RdRp) gene (14,94,95). Two variants of PCR, namely immunocapture RT-PCR (IC-RT-PCR) and multiplex-PCR (m-PCR), have been developed for the detection of various economically important viruses, including PMMoV (93,96). A novel single-tube multiplex IC-RT-PCR assay was developed, enabling the simultaneous detection of PMMoV and TMGMV (96). ...
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Viruses are one of the major restraining factors in pepper cultivation globally. Among different viruses, pepper mild mottle virus (PMMoV) is one of the most detrimental plant viruses infecting Capsicum spp. belonging to the genus Tobamovirus and Virgaviridae family. It has a monopartite positive-sense single-stranded RNA genome of 6.35 kb size. On an average, PMMoV results in 15%–40% losses in capsicum fruit yield. However, the incidence of PMMoV can reach as high as 95%, leading to substantial yield losses ranging from 75% to 95%. The virus is transmitted via contact, soil, and seeds rather than via insect vectors. PMMoV, because of its seed-borne nature, now occurs worldwide. PMMoV mainly infects Capsicum spp. under natural conditions; however, it can experimentally be transmitted to other plants species belonging to the families Solanaceae, Cucurbitaceae, Labiatae, Chenopodiaceae, and Plantaginaceae. The resistance to tobamoviruses in capsicum is conferred by L locus. Mutations in the coat protein of PMMoV are responsible for the emergence of L -mediated resistance-breaking pathotypes. The highly contagious nature of the virus, seed transmission behavior, and the emergence of virulence complicate its management through a single approach. Therefore, efforts are directed towards providing a more practical and efficient integrated management solution using the RNA interference approach; exploitation of the L gene for resistance breeding; and the inhibitory potential of natural products, systemic resistance-inducing antagonistic bacteria, and chemically synthesized silver nanoparticles. Markers linked to L alleles have been observed to accelerate capsicum breeding programs through marker-assisted selection. In this study, an attempt has been made to compile the recent developments in PMMoV biology, pathogenic variability, genomic organization, and management strategies.
... Therefore, it can lead to contamination in seed production and export. Since there was no antibody for this virus available in Thailand then, polyclonal and monoclonal antibodies were produced from PMMoV antigen and rapid test kits were developed for this virus and 4 pepper-infecting tobamoviruses (Phatsaman et al. 2020). Begomoviruses were not reported in the 1989-1991 survey which might be due to no evidence of symptoms associated with this pathogen observed in the production fields. ...
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Incidence and distribution of plant viruses infecting peppers in Thailand were surveyed from 2014-2016 to obtain updated information to support development of effective diagnosis and disease management. Field surveys for virus infection were conducted in 15 provinces encompassing 40 pepper-growing locations. Total numbers of 1,319 samples from 33 varieties were tested for virus infection using enzyme-linked immunosorbent assay with locally produced and commercial antibodies specific to chilli veinal mottle virus (ChiVMV), cucumber mosaic virus (CMV), potato virus Y (PVY), tobacco mosaic virus (TMV), tomato leaf curl New Delhi virus (ToLCNDV), tomato necrotic ring virus (TNRV), watermelon silver mottle virus (WSMoV) and pepper mild mottle virus (PMMoV). Of this sample number, 567 were positive to at least one pepper virus. Mean disease incidence (DI) indicated the highest value in the northern (93.41±8.62) followed by northeastern (92.60±9.99) and central (73.80±25.04) regions. The highest DI was in Uttaradit, Phitsanulok and Surin provinces (100%). Overall identified virus incidence was 42.99% whereas the highest value was in Chiang Rai province (72.31%). CMV was the major species among 3 surveyed regions (29.11%) followed by ChiVMV (11.83%), tospovirus serogroup IV (6.67%), PMMoV (5.53%), ToLCNDV (1.21%), PVY (1.14%), TMV (0.53%) and TNRV (0.45%). Mean disease severity showed the highest value in the northeastern (3.00±0.58) followed by northern (2.55±0.54) and central (2.05±0.86) regions. The Yok Siam pepper variety was found to be very susceptible to all viruses examined. Among mixed infections, incidence rate of ChiVMV + CMV was the highest at 41.8%. The information from this research provides useful information to support development of effective disease diagnosis and management for peppers in Thailand.
... Polyclonal antibodies were also developed non-commercially for the detection of Tobamoviruses. The antibodies developed for Pepper mild mottle virus (PMMoV) specifically detected another four Tobamoviruses (including ToMV) in an antibody dilution of 1:1000 and infected plant sap dilution of 1:5120 [48]. The polyclonal antibody developed for the detection of ToMV exhibited twice as high sensitivity in ELISA testing as commercial antibodies [49]. ...
Article
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Plant viruses threaten agricultural production by reducing the yield, quality, and economical benefits. Tomato mosaic virus (ToMV) from the genus Tobamovirus causes serious losses in the quantity and quality of tomato production. The management of plant protection is very difficult, mainly due to the vector-less transmission of ToMV. Resistant breeding generally has low effectiveness. The most practical approach is the use of a rapid diagnostic assay of the virus' presence before the symptoms occur in plants, followed by the eradication of virus-infected plants. Such approaches also include serological detection methods (ELISA and Western immunoblotting), where antibodies need to be developed for an immunochemical reaction. The development and characterization of polyclonal antibodies for the detection of ToMV with appropriate parameters (sensitivity, specificity, and cross-reactivity) were the subjects of this study. A new polyclonal antibody, AB-1, was developed in immunized rabbits using the modified oligopeptides with antigenic potential (sequences are revealed) derived from the coat protein of ToMV SL-1. the developed polyclonal antibody. AB-1, showed higher sensitivity when compared with commercially available analogs. It also detected ToMV in infected pepper and eggplant plants, and detected another two tobamoviruses (TMV and PMMoV) and ToMV in soil rhizosphere samples and root residues, even two years after the cultivation of the infected tomato plant.
... The inherent immunogenicity of a PVNP will likely lead to the development of an anti-PVNP immune response, both by B cells and by T cells. Anti-PVNP specific antibodies are demonstrated in response to administration of CPMV [49], alfalfa mosaic virus (AMV) [50], and pepper mild mottle virus (PMMoV) [51]. Interestingly, TMV nanoparticles camouflaged using serum albumin (SA) does not inhibit anti-TMV antibody production [52]. ...
Article
Plant virus nanoparticles (PVNPs) have inherent immune stimulatory ability, and have been investigated as immune adjuvants to stimulate an anti-tumor immune response. The combination of immune stimulation, nanoparticle structure and the ability to deliver other therapeutic molecules provides a flexible platform for cancer immunotherapy. Researching multifunctional PVNPs and their modification will generate novel reagents for cancer immunotherapy. Here we review the properties of PVNPs, and their potential for clinical utilization to activate anti-tumor innate and lymphoid immune responses. PVNP have potential utility for cancer immunotherapy as vaccine adjuvant, and delivery systems for other reagents as mono immunotherapy or combined with other immunotherapies. This review outlines the potential and challenges in developing PVNPs as cancer immunotherapy reagents.
... As PMMoVs, they were inducing mild mottle in plants sampled in the field. Serological tests with two anti-PMMoV IgGs showed cross-reaction among them, similar to those obtained in other works [45,46]. In fact, these three viruses were genetically close in the phylogenetic tree made from tobamovirus genome sequences available on public databases. ...
Article
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Tobamoviruses are often referred to as the most notorious viral pathogens of pepper crops. These viruses are not transmitted by invertebrate vectors, but rather by physical contact and seeds. In this study, pepper plants displaying mild mottle and mosaic symptoms were sampled in four different regions of Peru. Upon double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) tests, seven samples cross-reacted weakly with antibodies against pepper mild mottle virus (PMMoV), suggesting the presence of tobamoviruses. When employing RT-PCR, conserved primers amplified cDNA fragments of viruses from two putative new tobamovirus species in the samples. The complete genome of two representative isolates were, therefore, sequenced and analysed in silico. These viruses, which were tentatively named yellow pepper mild mottle virus (YPMMoV) and chilli pepper mild mottle virus (CPMMoV), shared highest nucleotide genome sequence identities of 83 and 85% with bell pepper mottle virus (BpeMV), respectively. Mechanical inoculation of indicator plants with YPMMoV and CPMMoV isolates did not show any obvious differences in host ranges. These viruses were also inoculated mechanically on pepper plants harbouring different resistance L alleles to determine their pathotypes. Pepper plants carrying unfunctional L alleles (L0) to tobamoviruses were infected by all isolates and presented differential symptomatology for YPMMoV and CPMMoV. On the other hand, pepper plants carrying L1, L2, L3 and L4 alleles were resistant to all isolates, indicating that these viruses belong to pathotype P0.
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