Development of polyclonal antisera against movement proteins of the three poleroviruses infecting cucurbits and their serological relationships

Background: Cucurbit aphid-borne yellows virus (CABYV), Melon aphid-borne yellows virus (MABYV) and Suakwa aphid-borne yellows virus (SABYV) are three critical viruses infecting cucurbit crops. The preparation of specific antiserum against the virus is crucial for both the detection of virus and understanding the functions of the related genes. However, there is no report about detecting the three viruses using antisera against movement proteins (MP). Methods: In this study, we constructed prokaryotic expression vectors of the three viral movement proteins and transferred them into Escherichia coli strain Rosetta to purify the fusion proteins. Then the polyclonal antisera were obtained by immunizing New Zealand white rabbits. Western blotting was used to demonstrate the applicability of the three antisera. Results: We discovered that the titer of antiserum against MP CABYV reached to 1: 512000, and the titers of antisera against MP MABYV and MP SABYV reached to 1:256000. The optimized working concentration range for the three antisera was from 1:10000 to 1:64000. Both antisera against MP CABYV and MP MABYV could only react with the corresponding MP. The antiserum against MP SABYV not only had the strongest reaction with its MP but also could react with MP CABYV and MP MABYV at relative weaker levels and all the three antisera had no serological reactions with other poleroviruses tested. Furthermore, our results showed that the three antisera could specifically detect movement proteins both in Nicotiana benthamiana and cucumber leaves. Conclusions: We have established a sensitive system for detecting three poleroviruses infecting cucurbits by antisera against movement proteins, providing a material foundation for the future research on both the serological detection of viruses and the interaction mechanisms between the virus and host plants.