A sequel study on the occurrence of Tomato spotted wilt virus (TSWV) in cut-chrysanthemum by DAS-ELISA using recombinant nucleocapsid protein to produce polyclonal antiserum

Abstract

The tomato spotted wilt virus (TSWV) belonging to the genus Orthotospovirus, family Tospoviridae, causes severe necrotic disease in field crops and horticultural crops, resulting in considerable yield loss worldwide. The development of protein-based diagnostics is essential to track the virus transmission and prevent its spread in vegetatively propagated crops such as ornamentals. In this study, nucleocapsid (N) gene of TSWV was cloned in pET 28 a (+) expression vector. Expression of the 32 kDa recombinant TSWV-N protein was induced in BL21 (DE3) cells using 1 mM of Isopropyl β-d-1-thiogalactopyranoside (IPTG), and was confirmed through SDS-PAGE and Western blot by fluorescent-labeled secondary antibody. The bacterial cells expressed recombinant TSWV-N protein up to a concentration of 9.48 μg/mL. The purified protein was used for immunization of a rabbit to produce specific polyclonal antiserum. The TSWV antiserum was conjugated with the enzyme alkaline phosphatase (ALP). Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA) was developed and validated against TSWV infected hosts. This antiserum specifically reacted with recombinant N protein as well as TSWV infected hosts, but not with groundnut bud necrosis orthotospovirus (GBNV) as well as capsicum chlorosis orthotospovirus (CaCV) infecting tomato and chilli plants. The coating antibody at 1 μg/mL concentration and 1:500 dilution of enzyme conjugate were found to be effective and economical in the detection of recombinant N protein of TSWV and the virus present naturally in the infected hosts. Using standardized DAS-ELISA protocol, the TSWV titer also was quantified in artificially inoculated assay hosts. Among 11 hosts tested, higher virus titer was recorded in Nicotiana tabacum (0.270 μg/100 μL), followed by Impatiens balsamiana (0.185 μg/100 μL) and Dahlia pinnata at a low virus tire of 0.083 μg/100 μL. The diagnostic reagents and protocol (DAS-ELISA) are further validated by detecting the infection of TSWV in chrysanthemum stem cuttings from six different nurseries in the hill stations of Tamil Nadu, India. The DAS-ELISA assay experimented on six varieties from four different nurseries revealed that the Mum Yellow variety had a higher percentage of TSWV infection (36 %), which was followed by the Mum White variety (33 %); both collected from Kotagiri Nursery. The same variety exhibited a higher virus titer by DAS-ELISA, an A405 value range of 0.733 (̴ 0.115 μg) and 0.711 (̴ 0.111 μg) respectively, and a total of 27 % of TSWV infection was confirmed by screening 800 stem cuttings by DAS-ELISA. The presence of TSWV was also detected in 54 (6.75 %) asymptomatic stem cuttings from different locations, and the A405 value ranged from 0.325 to 0.468.(̴ 0.044−0.069 μg/100 μL); this is the first reported development of immune-based diagnostics for TSWV in India. This protocol and diagnostics will be highly useful for quarantine purposes while trading large quantities of planting materials.