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The reprogramming of host physiology has been considered an essential process for baculovirus propagation. Trehalose, the main sugar in insect blood, plays a crucial role as an instant energy source. Although the trehalose level is modulated following infection with Bombyx mori nucleopolyhedrovirus (BmNPV), the mechanism of trehalose metabolism in response to BmNPV infection is still unclear. In this study, we demonstrated that the trehalose level tended to be lower in BmNPV-infected hemolymph and higher in the midgut. The omics analysis revealed that two trehalose transporters, BmTret1-1 and BmTret1-2, and trehalase, BmTRE1 and BmTRE2, were differentially expressed in the midgut after BmNPV infection. BmTret1-1 and BmTret1-2 had the ability to transport trehalose into the cell and promoted cellular absorption of trehalose. Furthermore, the functions of BmTret1-1, BmTret1-2, BmTRE1 and BmTRE2 in BmNPV infection were analyzed. These genes were upregulated in the midgut after BmNPV infection. Virus amplification analysis revealed that these genes could promote BmNPV proliferation in BmN cells. In addition, these genes could promote the expression of BmPI3K, BmPDK1 and BmAkt and inhibit the expression of BmFoxO in the phosphoinositide 3-kinase (PI3K)-Akt signalling pathway. Similarly, the increased trehalose level in BmN cells could promote the expression of BmPI3K, BmPDK1 and BmAkt and inhibit the expression of BmFoxO. Taken together, BmNPV infection promote the expression of trehalose hydrolysis and transport-related genes. These changes affect the PI3K-Akt signalling pathway to facilitate BmNPV proliferation. These findings help clarify the relationship between trehalose metabolism and BmNPV infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious pathogenic microorganism that causes tremendous loss to sericulture. Previous studies have found that some proteins of serine protease family in the digestive juice of B. mori larvae have anti-BmNPV activity. In our previous publication about proteome analysis of the digestive juice of B. mori larvae, the digestive enzyme trypsin, alkaline A (BmTA) was filtered as a differentially expressed protein possibly involved in BmNPV resistance. Here, the biological characteristics and anti-BmNPV functions of BmTA were comprehensively analysed. The cDNA sequence of BmTA had an ORF of 768 nucleotides encoding 255 amino acid residues. Domain architecture analysis showed that BmTA contained a signal peptide and a typical Tryp_SPc domain. Quantitative real-time PCR analysis showed that BmTA was highly expressed in the larval stages and specifically expressed in the midgut of B. mori larvae. The expression level of BmTA in BmNPV resistant strain A35 was higher than that in susceptible strain P50. After BmNPV infection, the expression of BmTA increased in both strains from 24 to 72 h. Virus amplification analysis showed that the relative levels of VP39 in B. mori larvae and BmN cells infected with the appropriate concentration of recombinant-BmTA-treated BmNPV were significantly lower than in the control groups. Moreover, overexpression of BmTA in BmN cells significantly inhibited the amplification of BmNPV. Taken together, the results of this study indicated that BmTA possessed anti-BmNPV activity in B. mori, which broadens the horizon for virus-resistant breeding of silkworms.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity. In this study, antiviral activity examination of different resistant strains showed that the digestive juice of the resistant strain (A35) had higher inhibition to virus than the susceptible strain (P50). Subsequently, the label-free quantitative proteomics was used to study the midgut digestive juice response to BmNPV infection in P50 and A35 strains. A total of 98 proteins were identified, of which 80 were differentially expressed proteins (DEPs) with 54 enzymes and 26 nonenzymatic proteins by comparing the proteomes of infected and non-infected P50 and A35 silkworms. These DEPs are mainly involved in metabolism, proteolysis, neuroactive ligand receptor interaction, starch and sucrose metabolism and glutathione metabolism. After removing the genetic background and individual immune stress response proteins, 9 DEPs were identified potentially involved in resistance to BmNPV. Further studies showed that a serine protease, an alkaline phosphatase and serine protease inhibitor 2 isoform X1 were differentially expressed in A35 compared to P50 or post BmNPV infection. Taken together, these results provide insights into the potential mechanisms for silkworm digestive juice to provide resistance to BmNPV infection. Signifcance: Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, which has a great impact on the sericulture. BmNPV entered the midgut lumen and exposed to digestive juices after oral infection. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity, however, current information on the digestive juice proteome of high resistant silkworm strain after BmNPV challenge compared to susceptible strain is incomprehensive. Here, we combined label-free quantification method, bioinformatics, RT-qPCR and western blot analysis and found that BmNPV infection causes some protein changes in the silkworm midgut digestive juice. The DEPs were identified in the digestive juices of different resistant strains following BmNPV infection, and screened out some proteins potentially related to resistance to BmNPV. Three important differentially expression proteins were validated by independent approaches. These findings uncover the potential role of silkworm digestive juice in providing resistance to BmNPV and supplemented the profile of the proteome of the digestive juices in B. mori., Graphical abstract Unlabelled Image, Highlights • The digestive juice antiviral activity of resistant strain was higher than susceptible strain. • Silkworm digestive juice was analyzed by label-free quantitative proteomics. • Eighty DEPs were identified in different resistant strains following BmNPV infection. • Some DEPs were validated by RT-qPCR and western blot. • These results provide insights into the potential mechanisms for B. mori resistant to BmNPV infection.