Isolation and Purification of CSBV

For each of the three strains (LNQY-2008: ~70.0% proportion of the infected larvae, SXYL-2015: ~80.0% proportion of the infected larvae, and JLCBS-2014: ~73.3% proportion of the infected larvae), 50 infected A. cerana larvae were collected. After weighing, larvae were completely homogenised in sterile water (1.5-fold amount, by weight) using a pestle and mortar. CSBV purification was performed by cesium chloride gradient centrifugation, according to Ma’s method12 (link)32 (link). The supernatant was then passed through a 0.45-μm cell filter first and then through a 0.22-μm cell filter. The filtrate was then fed to the second instar of A. cerana larvae for virus passage, and 50 diseased larvae were taken from each culture plate after 8 days. The virus was isolated and purified again by the above method. Next, virus suspension was analyzed by the RT-PCR method31 (link) for the following viruses: black queen cell virus (BQCV)33 (link), acute bee paralysis virus (ABPV)33 (link), chronic bee paralysis virus (CBPV)34 (link), deformed wing virus (DWV)34 (link), kashmir bee virus (KBV)35 (link), Israeli acute paralysis virus (IAPV)36 (link), and CSBV31 (link). Three virus suspensions, after we proved that they did not contain other viruses in addition to CSBV, were stored at −80 °C until use.