Screening of PI3K-Akt-targeting Drugs for Silkworm against Bombyx mori Nucleopolyhedrovirus

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most prevalent threat to silkworms. Hence, there is a need for antiviral agents in sericulture. The PI3K-Akt pathway is essential for the efficient replication of the baculovirus. In an attempt to screen antiviral drugs against BmNPV, we summarized the commercial compounds targeting PI3K-Akt and selected the following seven oral drugs for further analyses: afuresertib, AZD8835, AMG319, HS173, AS605240, GDC0941, and BEZ235. Cell viability assay revealed that the cytotoxicity of these drugs at 10 µM concentration was not strong. Viral fluorescence observation and qPCR analysis showed that these candidate drugs significantly inhibited BmNPV in BmE cells. Only AMG319 and AZD8835 inhibited viral proliferation in silkworm larvae. The mortality of AZD8835-treated silkworms was lower than that of the control silkworms. Western blotting showed that AMG319 and AZD8835 decreased p-Akt expression after BmNPV infection. These results suggest that AZD8835 has application potential in sericulture.


Introduction
The silkworm is an important economic insect for silk production. Sericulture is one of the main sources of income for farmers in several developing countries [1,2]. However, silkworm diseases cause serious economic losses in sericulture. Viruses including Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), B. mori densovirus (BmDNV), and B. mori nucleopolyhedrovirus (BmNPV), in particular, pose the most serious threat to silkworms [1]. Therefore, there is a need for antiviral agents in sericulture.
Breeding antiviral silkworm strains using transgenic technology is an approach employed for virus control [1]. For instance, overexpression of Bmlipase-1 inhibits BmNPV during the initial infection stage [2]. Silencing of viral genes by RNAi suppresses the viral mRNA [3][4][5]. Overexpression of hycu-ep32 inhibits viral protein synthesis [6]. Regulation of host immunity via overexpression of PGRPs enhances the antiviral capacity of silkworms [1,7]. Simultaneous overexpression of antiviral genes and knock-down of viral genes improves the resistance of transgenic silkworms [8]. Transgenic expression of Cas9 and gRNAs targeting the BmNPV genes also inhibits BmNPV in silkworms [9]. A safety assessment of transgenic silkworms has to be performed before commercialization. Antiviral drugs have been developed for coping with viral infection. Five compounds targeting viral proteins, namely, rimantadine, amantadine, zanamivir, oseltamivir, and baloxavir marboxil, have been approved by the FDA for inhibiting influenza virus [10,11]. Some host factors are involved in viral

Analysis of Cytotoxicity of Candidate Drugs
In order to reduce costs, low doses of drugs are required to inhibit viruses in sericulture. Referring to the concentrations of these drugs used in other studies, 10 µM of each drug was chosen for detection in BmE cells. The viability of BmE cells treated with 10 µM AMG319, AZD8835, afuresertib, GDC0941, AS605240, BEZ235, HS173, and LY294002 was 92%, 83%, 84%, 86%, 92%, 80%, 107%, and 86%, respectively, compared with that of the cells treated with the DMSO control ( Figure 1A), suggesting that the candidate drugs at 10 µM concentration do not have a strong cytotoxic effect. The cytotoxicity of AZD8835 in BmE cells was low upon treatment with 100 µM ( Figure 1B). 107%, and 86%, respectively, compared with that of the cells treated with the DMSO control ( Figure  1A), suggesting that the candidate drugs at 10 µM concentration do not have a strong cytotoxic effect. The cytotoxicity of AZD8835 in BmE cells was low upon treatment with 100 µM ( Figure 1B).

Candidate Drugs Inhibited BmNPV in BmE Cells
The virus fluorescence in drug-treated cells was obviously weaker than that in the control cells ( Figure 2). The qPCR results showed that the drugs significantly inhibited BmNPV multiplication, and the accumulated virus DNA in AMG319-, AZD8835-, afuresertib-, GDC0941-, AS605240-, BEZ235-, HS173-, and LY294002-treated cells was 60%, 42%, 35%, 19%, 14%, 24%, 3%, and 36% compared with that of the DMSO-treated cells, respectively ( Figure 3). These results revealed that the candidate drugs can inhibit BmNPV in BmE cells. 107%, and 86%, respectively, compared with that of the cells treated with the DMSO control ( Figure  1A), suggesting that the candidate drugs at 10 µM concentration do not have a strong cytotoxic effect. The cytotoxicity of AZD8835 in BmE cells was low upon treatment with 100 µM ( Figure 1B).

AMG319 and AZD8835 Decreased p-Akt after BmNPV Infection
Western blotting showed that the p-Akt level was decreased in AMG319-and AZD8835-treated BmE cells when compared with that in the DMSO-treated control cells ( Figure 5). A band of control GAPDH was detected, whereas the band of p-Akt was not observed in the total protein extracted from whole silkworms (data not shown). These results suggest that AMG319 and AZD8835 inhibit p-Akt after virus infection.

Discussion
The PI3K-Akt pathway is required for the efficient replication of baculovirus [12,16]. BmNPV infects silkworm mostly via the oral route [1]. In this study, seven commercial oral drugs targeting PI3K-Akt were selected to determine their cytotoxicity and inhibitory effect on BmNPV. Among these, AZD8835 exhibited the strongest antiviral effect in BmE cells and silkworm larvae.
Studies have reported that AcMNPV and BmNPV induced cellular p-Akt, and LY294002 inhibited induced p-Akt and viral proliferation in Sf9 and BmE cells [12,16]. The results of the present study show that LY294002 inhibits BmNPV in BmE cells (Figure 3) but not in silkworm larvae ( Figure  4), as it is an intravenous drug. AMG319 and AZD8835 inhibited p-Akt in virus-infected BmE cells ( Figure 5), but it was unexpected that p-Akt was not detected in whole silkworms. Further analysis revealed that p-Akt was detected in multiple tissues, but not in whole fifth instar larvae (data not shown). It is presumed that mulberry leaf consumption in whole silkworm affects the detection of p-Akt. Afuresertib, GDC0941, BEZ235, and HS173 significantly inhibited BmNPV in BmE cells ( Figure  3). However, the viral content was increased after oral administration of these drugs in silkworms (Figure 4), which might be caused by drug toxicity to larval individuals. Differences in intestinal pH between insects and mammals might affect drug absorption and even cause drug toxicity.
AZD8835 presented the strongest inhibitory effect on BmNPV among all the candidate drugs, and it reduced the mortality of silkworms to 16% after infection compared with that of the control (Figure 4). Whether AZD8835 can inhibit BmCPV and BmDNV in silkworms needs to be verified in the future. To develop drugs with higher antiviral capacity that could be applied in sericulture, the following aspects should be considered in future studies. First, optimize the structure of drugs to adapt to the digestion and absorption system of insects, which might increase absorption efficiency and decrease cytotoxicity [31]. Second, use carriers to promote the utilization of drugs.

AMG319 and AZD8835 Decreased p-Akt after BmNPV Infection
Western blotting showed that the p-Akt level was decreased in AMG319-and AZD8835-treated BmE cells when compared with that in the DMSO-treated control cells ( Figure 5). A band of control GAPDH was detected, whereas the band of p-Akt was not observed in the total protein extracted from whole silkworms (data not shown). These results suggest that AMG319 and AZD8835 inhibit p-Akt after virus infection.

AMG319 and AZD8835 Decreased p-Akt after BmNPV Infection
Western blotting showed that the p-Akt level was decreased in AMG319-and AZD8835-treated BmE cells when compared with that in the DMSO-treated control cells ( Figure 5). A band of control GAPDH was detected, whereas the band of p-Akt was not observed in the total protein extracted from whole silkworms (data not shown). These results suggest that AMG319 and AZD8835 inhibit p-Akt after virus infection.

Discussion
The PI3K-Akt pathway is required for the efficient replication of baculovirus [12,16]. BmNPV infects silkworm mostly via the oral route [1]. In this study, seven commercial oral drugs targeting PI3K-Akt were selected to determine their cytotoxicity and inhibitory effect on BmNPV. Among these, AZD8835 exhibited the strongest antiviral effect in BmE cells and silkworm larvae.
Studies have reported that AcMNPV and BmNPV induced cellular p-Akt, and LY294002 inhibited induced p-Akt and viral proliferation in Sf9 and BmE cells [12,16]. The results of the present study show that LY294002 inhibits BmNPV in BmE cells ( Figure 3) but not in silkworm larvae ( Figure  4), as it is an intravenous drug. AMG319 and AZD8835 inhibited p-Akt in virus-infected BmE cells ( Figure 5), but it was unexpected that p-Akt was not detected in whole silkworms. Further analysis revealed that p-Akt was detected in multiple tissues, but not in whole fifth instar larvae (data not shown). It is presumed that mulberry leaf consumption in whole silkworm affects the detection of p-Akt. Afuresertib, GDC0941, BEZ235, and HS173 significantly inhibited BmNPV in BmE cells ( Figure  3). However, the viral content was increased after oral administration of these drugs in silkworms (Figure 4), which might be caused by drug toxicity to larval individuals. Differences in intestinal pH between insects and mammals might affect drug absorption and even cause drug toxicity.
AZD8835 presented the strongest inhibitory effect on BmNPV among all the candidate drugs, and it reduced the mortality of silkworms to 16% after infection compared with that of the control (Figure 4). Whether AZD8835 can inhibit BmCPV and BmDNV in silkworms needs to be verified in the future. To develop drugs with higher antiviral capacity that could be applied in sericulture, the following aspects should be considered in future studies. First, optimize the structure of drugs to adapt to the digestion and absorption system of insects, which might increase absorption efficiency and decrease cytotoxicity [31]. Second, use carriers to promote the utilization of drugs.

Discussion
The PI3K-Akt pathway is required for the efficient replication of baculovirus [12,16]. BmNPV infects silkworm mostly via the oral route [1]. In this study, seven commercial oral drugs targeting PI3K-Akt were selected to determine their cytotoxicity and inhibitory effect on BmNPV. Among these, AZD8835 exhibited the strongest antiviral effect in BmE cells and silkworm larvae.
Studies have reported that AcMNPV and BmNPV induced cellular p-Akt, and LY294002 inhibited induced p-Akt and viral proliferation in Sf9 and BmE cells [12,16]. The results of the present study show that LY294002 inhibits BmNPV in BmE cells ( Figure 3) but not in silkworm larvae (Figure 4), as it is an intravenous drug. AMG319 and AZD8835 inhibited p-Akt in virus-infected BmE cells ( Figure 5), but it was unexpected that p-Akt was not detected in whole silkworms. Further analysis revealed that p-Akt was detected in multiple tissues, but not in whole fifth instar larvae (data not shown). It is presumed that mulberry leaf consumption in whole silkworm affects the detection of p-Akt. Afuresertib, GDC0941, BEZ235, and HS173 significantly inhibited BmNPV in BmE cells ( Figure 3). However, the viral content was increased after oral administration of these drugs in silkworms (Figure 4), which might be caused by drug toxicity to larval individuals. Differences in intestinal pH between insects and mammals might affect drug absorption and even cause drug toxicity.
AZD8835 presented the strongest inhibitory effect on BmNPV among all the candidate drugs, and it reduced the mortality of silkworms to 16% after infection compared with that of the control (Figure 4). Whether AZD8835 can inhibit BmCPV and BmDNV in silkworms needs to be verified in the future. To develop drugs with higher antiviral capacity that could be applied in sericulture, the following aspects should be considered in future studies. First, optimize the structure of drugs to adapt to the digestion and absorption system of insects, which might increase absorption efficiency and decrease cytotoxicity [31]. Second, use carriers to promote the utilization of drugs. Polyamidoamine can be administered orally and stably in mice, which can improve the utilization of oral drugs [32].
Third, reduce the cost of drugs by controlling the purity of drugs and standards of the production process. Last, enhance the inhibitory effect of drugs on viruses by targeting multiple host factors, such as ERK, JNK [33], and PI3K-Akt, which are identified to be important for virus infection.
In summary, we evaluated the inhibitory ability of seven oral drugs from the range of commercial PI3K-Akt-targeting drugs against BmNPV. AZD8835 inhibits viral proliferation in BmE cells and silkworm larvae after infection of BmNPV, indicating its potential application to decrease mortality in sericulture after further optimization.