Dataset Information

Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in Escherichia coli.

ABSTRACT: A Korean isolate of the sacbrood virus infecting Apis cerana (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in Escherichia coli BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 10³ copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 10⁴ copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.

SUBMITTER: Lee SH

PROVIDER: S-EPMC8707083 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:We determined the complete genome sequence of a sacbrood virus (SBV) infecting Indian honey bee (Apis cerana indica) from Tamil Nadu, India named as AcSBV-IndTN1. The genome of AcSBV-IndTN1 comprised of 8740 nucleotides, encoding a single large ORF containing 2849 amino acids flanked by 5' and 3' untranslated regions. Results of phylogenetic tree analysis based on complete genomes of SBV isolates indicated that the virus isolates from India isolated from the Asiatic honey bee A. cerana (AcSBVs) formed a separate group along with six Vietnam isolates and three Chinese isolates. The AcSBV-IndTN1 isolate showed closer genetic relationship with other isolates from India. The second major group had both AcSBVs and AmSBVs (virus isolated from European honey bee, Apis mellifera SBV) of Korea, China and Vietnam. The third and a distantly related group had AmSBVs of Australia, UK, USA and Korea. The results obtained from phylogenetic analysis were further supported with evolutionary distance analysis. AcSBV-IndTN1 isolate open reading frame had 95-99% amino acid sequence similarity with other Indian isolates and 92-96% with AcSBVs and AmSBVs of other geographical locations. In addition, sequence difference count matrix ranged from 154 to 907 nt among all the SBV isolates. This suggests that the virus isolates have evolved significantly in different geographical locations but isolates on different hosts in a given location/country are closely related. The high similarity in the genome among the AcSBV and AmSBV isolates indicate possible cross-infections and recombination of SBV isolates in Asian continent where both the honey bee species are reared in close proximity. Gene flow between SBV population indicating that an infrequent gene flow occur between them. The pattern of molecular diversity in SBV population revealed that the occurrence of recent population expansion of SBV. To the best of our knowledge this is the first report of the complete nucleotide sequence of AcSBV from Tamil Nadu, India. This study provided an opportunity to establish the molecular evolution of SBV isolates and shall be useful in the development of diagnostics and effective disease control strategies.

Dataset Information

Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets