Identification of cuticular protein genes and analysis of their roles in phosphine resistance of the rusty grain beetle Cryptolestes ferrugineus.

The rusty grain beetle, Cryptolestes ferrugineus (Stephens) is one of the most economically important stored grain pests, and it has evolved the high resistance to phosphine. Cuticular proteins (CPs) are the major structural components of insect cuticle, and previous studies have confirmed that CPs were involved in insecticide resistance. However, the CPs of C. ferrugineus are still poorly characterized, and thus we conducted transcriptome-wide identification of CP genes and analyze their possible relationships with phosphine resistance in this pest. In this study, a total of 122 putative CPs were annotated in the C. ferrugineus transcriptome data by blasting with the known CPs of Tribolium castaneum. The analysis of conserved motifs revealed these CPs of C. ferrugineus belonging to 9 different families, including 87 CPR, 13 CPAP1, 7 CPAP3, 3 Tweedle, 1 CPLCA, 1 CPLCG, 5 CPLCP, 2 CPCFC, and 3 CPFL proteins. The further phylogenetic analysis showed the different evolutionary patterns of CPs. Namely, we found some CPs (CPR family) formed species-specific protein clusters, indicating these CPs might occur independently among insect taxa, and while some other CPs (CPAP1 and CPAP3 family) shared a closer correlation based on the architecture of protein domains. Subsequently, the previous RNA-seq data were applied to establish the expression profiles of CPs in a phosphine susceptible and resistant populations of C. ferrugineus, and a large amount of CP genes were found to be over-expressed in resistant insects. Lastly, an up-regulated CP gene (CPR family) was selected for the further functional analysis, and after this gene was silenced via RNA interference (RNAi), the sensitivity to phosphine was significantly enhanced in C. ferrugineus. In conclusion, the present results provided us an overview of C. ferrugineus CPs, and which suggested that the CPs might play the critical roles in phosphine resistance.

Myricetin inhibits pseudorabies virus infection through direct inactivation and activating host antiviral defense.

Myricetin, a polyhydroxyflavone compound, is one of the main ingredients of various human foods and therefore also known as dietary flavonoids. Due to the continuous emergence of resistant strains of herpesviruses, novel control measures are required. In the present study, myricetin exhibited potent antiviral activity against pseudorabies virus (PRV), a model organism of herpesvirus. The suppression rate could reach up to 96.4% at a concentration of 500 µM in cells, and the 50% inhibitory concentration (IC50) was 42.69 µM. Moreover, the inhibitory activity was not attenuated by the increased amount of infective dose, and a significant reduction of intracellular PRV virions was observed by indirect immunofluorescence. A mode of action study indicated that myricetin could directly inactivate the virus in vitro, leading to inhibition of viral adsorption, penetration and replication in cells. In addition to direct killing effect, myricetin could also activate host antiviral defense through regulation of apoptosis-related gene expressions (Bcl-2, Bcl-xl, Bax), NF-κB and MAPK signaling pathways and cytokine gene expressions (IL-1α, IL-1ß, IL-6, c-Jun, STAT1, c-Fos, and c-Myc). In PRV-infected mouse model, myricetin could enhance the survival rate by 40% at 5 days post infection, and viral loads in kidney, liver, lung, spleen, and brain were significantly decreased. The pathological changes caused by PRV infection were improved by myricetin treatment. The gene expressions of inflammatory factors (MCP-1, G-CSF, IL-1α, IL-1ß, and IL-6) and apoptotic factors (Bcl-xl, Bcl-2, and Bax) were regulated by myricetin in PRV-infected mice. The present findings suggest that myricetin can effectively inhibit PRV infection and become a candidate for development of new anti-herpesvirus drugs.

Antiviral effect of an extract from Kaempferia galanga L. rhizome in mice infected with pseudorabies virus.

Pseudorabies virus (PrV) is one of the most important herpesviruses which can cause severe diseases in many mammals and some avian species. In recent years, repeated outbreaks of pseudorabies worldwide indicated an urgent need for new control measures. The results described in this study demonstrated that an extract prepared from the rhizome of Kaempferia galanga L (Kge), which consisted of flavonoids (2.82%), saccharides (61.37%), phenols (1.22%) and saponins (3.10%), possessed a potent anti-PrV activity. In PK-15 cells, Kge treatment inhibited PrV-induced cell death by more than 90% at a dose of 200 µg/mL. The 50% inhibitory concentration (IC50) was 55.85 µg/mL. In the PrV-infected mice treated with Kge, the survival rate was up to 60% at day 6 post-infection, while the infected mice without Kge treatment all died. The virus titers in the brains of the Kge-treated infected mice were significantly reduced. Kge treatment also alleviated the severity of the PrV-induced lesions in the heart, liver, spleen, lung and kidney. Kge exhibited immune-regulating activity through the regulation of cytokines (IFN-α, IFN-ß, IL-4, IL-6 and TNF-α) in the serum of PrV-infected mice, suggesting that one possible mechanism of anti-PrV activity was through the regulation of immune function. These results suggested that Kge could be a promising drug candidate for treating PrV infections.

The antiviral activity of kaempferol against pseudorabies virus in mice.

BACKGROUND: Pseudorabies virus (PRV), a member of the Alphaherpesviruses, is one of the most important pathogens that harm the global pig industry. Accumulated evidence indicated that PRV could infect humans under certain circumstances, inducing severe clinical symptoms such as acute human encephalitis. Currently, there are no antiviral drugs to treat PRV infections, and vaccines available only for swine could not provide full protection. Thus, new control measures are urgently needed. RESULTS: In the present study, kaempferol exhibited anti-PRV activity in mice through improving survival rate by 22.22 %, which was higher than acyclovir (Positive control) with the survival rate of 16.67 % at 6 days post infection (dpi); meanwhile, the survival rate was 0 % at 6 dpi in the infected-untreated group. Kaempferol could inhibit the virus replication in the brain, lung, kidney, heart and spleen, especially the viral gene copies were reduced by over 700-fold in the brain, which was further confirmed by immunohistochemical examination. The pathogenic changes induced by PRV infection in these organs were also alleviated. The transcription of the only immediate-early gene IE180 in the brain was significantly inhibited by kaempferol, leading to the decreased transcriptional levels of the early genes (EPO and TK). The expression of latency-associated transcript (LAT) was also inhibited in the brain, which suggested that kaempferol could inhibit PRV latency. Kaempferol-treatment could induce higher levels of IL-1ß, IL-4, IL-6, TNF-α and IFN-γ in the serum at 3 dpi which were then declined to normal levels at 5 dpi. CONCLUSIONS: These results suggested that kaempferol was expected to be a new alternative control measure for PRV infection.