Genetic And Phylogenetic Analysis On Honey Bee Sacbrood Virus Isolates And The Differences Of Immunity And Gut Microbiota Between Apis Mellifera And Apis Cerana Infected With Sacbrood Virus

Sacbrood virus(SBV)is one of the most common pathogens that infect honeybees and pose a serious threat to honeybee health.However,SBV is more lethal to Apis cerana than Apis mellifera.Apis cerana and Apis mellifera have different mechanisms to deal with virus infection.The aim of this study was to investigate the genetic diversity of SBV and the changes of immune regulation,nutritional metabolism,antiviral and gut microbiota between Apis cerana and Apis mellifera after SBV infection.In this experiment,SBV virus infection in Nanchang,Zixi,Tonggu,Yifeng and Jinxi in Jiangxi province was detected by RT-PCR.SBV fragment for phylogenetic analysis was amplified from the samples detected with the SBV.The amplification products were directly sequenced,then the phylogenetic tree was constructed and the phylogenetic relationship of SBV was analyzed.The results indicated that SBV was detected only in Zixi and Tonggu,the detection rate was 20%.Zixi and Tonggu isolates in this study are genetically closely related to Chinese strains.Furthermore,the host specificity and geographic region significantly influence the phylogeny of SBV isolates.In order to explore the difference of SBV resistance mechanism between Apis cerana and Apis mellifera,we raised 2-day-old larvae of Apis cerana and Apis mellifera under laboratory conditions,inoculated them with SBV at the 3-day-old age,and sampled them on the first day after infection(4-day-old)and at the metagenesis stage(7-day-old).During the feeding period,the death rate of larvae was calculated daily and the survival curve was plotted.The relative expression levels of SBV and immunity,nutrition and antiviral genes in the two bee species at different age were detected by q PCR.The results indicated that the survival rate of Apis cerana larvae infected with SBV lowerthan that of the control group,the survival rate of Apis mellifera was not significantly different from the control group,and the relative expression of SBV in Apis cerana larvae at the same age was also significantly higher than that of Apis mellifera.These results further confirmed that Apis cerana was more sensitive to SBV than Apis mellifera,and the virulence of SBV was closely related to its infection level.After SBV infection,Apis cerana larvae rapidly produced a significant immune response,and the expression level of antimicrobial peptides increased rapidly.Nutritional metabolism and RNAi also responded and adjusted to deal with SBV infection.The expression level of antimicrobial peptides in Apis mellifera larvae decreased significantly,and the nutrient metabolism,cell composition and development genes were also widely down-regulated,showing a completely different defense mechanism from Apis cerana larvae.In addition,in order to understand the harm of SBV infection from the gut microbiota level,the 4-day-old and 7-day-old samples of Apis cerana and Apis mellifera larvae were sequenced using Illumina Nova Seq platform for amplification of 16 S r RNA gene V3-V4 region.The results indicated that SBV decreased the abundance of Bacillus in the7-day-old larvae of Apis cerana,while the abundance of Streptomyces and Brevundimonas of Apis mellifera larvae infected with SBV significantly increased.Furthermore,SBV decreased the gut microbiota diversity of the 7-day-old larvae of Apis cerana.There were significant differences in gut microbiota diversity between Apis cerana and Apis mellifera at the same age.Further studies showed that Bacillus Subtilis had a certain inhibitory effect on SBV.