| Nucleotide analogues are the main therapeutic drugs for herpes virus. The target of these drugs are similar, which may lead to cross resistance. Nowadays resistance strains already exist clinically. Therefore, searching for new target drugs is the key of future studies on herpes virus drugs. Duck plague virus (DPV) is an acute infectious virus which belongs to herpes virus family. Currently no therapeutic drug was found for the treatment on DPV. Only prevention with vaccination is usually taken. The study is to screen the anti-DPV active agents from natural medicinal products and research on the antiviral mechanism of the agents to find a new target of drugs and provide new methods on treating DPV. The study can also provide new targets and idea on the development of anti-herpes virus drugs. The research could be divided into the following parts:1. Screening of in-vitro anti-DPV active agents The research of in-vitro anti-DPV was carried out by CPE and MTT colorimetric methods on the basis of various natural medicinal products. Results are as follows:① In traditional herb crude extracts, alcohol extracts of Polygonum cuspidaturn and radix rubiae have some anti-virus activity:The inhibition ratio of Polygonum cuspidaturn alcohol extracts on DPV is 76.5%; the inhibition ratio of radix rubiae alcohol extracts on DPV is 48.91%.② The maximum inhibition ratios of petroleum ether extracts, ethyl acetate extracts and water extract attained from neem seed alcohol extract are 13.23%,29.24% and 11.72% respectively. ③ In cinnamomum longepaniculatum volatile oil, the inhibition ratios of Terpinen-4-O1,α-Terpinene, γ-Terpinene and 1.8-cineole are 4.42%,14.27%. 25.33% and 21.02% respectively. According to the above results, the anti-DPV effects of Polygonum cuspidatum is the best of all the natural medicinal products tested.2. Research of Resveratrol in-vitro anti-duck plague virus activity. Taking the main medicinal agents resveratrol of Polygonum cuspidatum as the studying object, the resveratrol in-vitro anti-DPV activity was tested via CPE, MTT and Titer methods. Results are as follow:① Observe the inhibition effect of resveratrol on DEFs lesion caused by DPV in the way of CPE method. As a result, the CPE can be completely inhibited when resveratrol concentration reaches 31.25μg/mL.② Estimate the inhibition effects of resveratrol on DPV with MTT method. It was found that the median inhibitory concentration of resveratrol in-vitro anti-DPV was 3.85±0.08μg/mL; median toxic concentration was 61.07±0.24 μg/mL; the therapeutic index was 15.86. ③ Use titer method to detect the reproduced DPV titers in DEFs with the influence of resveratrol. The results revealed that resveratrol can significantly lower the viral titer after its reproduction in cells. According to the above results, resveratrol has good in-vitro anti-DPV activity that deserves further studies.3. The establishment of TaqMan Absolute fluorescent quantitative PCR method and resveratrol’s influence on in-vitro replication Adopt DPV UL30 gene as the target fragment; design primer and probe; establish fluorescent quantitative PCR method. The results showed that the standard curvilinear relation (R2=0.997) established with this method has an amplification efficiency of 99.7%; it also has good specificity, sensitivity and stability. This method was applied to detect resveratrol’s growth influence on DPV in DEFs. The detection results are as following: ① The viral infection groups:Viral particles started to increase when DEFs was infected for 8h;it reached the peak at 48h. ② Resveratrol action groups:the copy number of viral particles had no significant increase within 24h; the copy number of viral particles started to rise after 24h and the rising speed was apparently lower than the viral infection groups (p<0.05). According to the above results, the incubation of DPV in DEFs can be extended by resveratrol and resveratrol has significant inhibition effects on its replication in host cells.4. The study of Resveratrol’s in-vitro anti-DPV feature To study the resveratrol anti-DPV feature with the following methods:use drug in various ways, medicate in different time, and use transmission electron microscope (TEM). The results are as following:① To investigate resveratrol’s in-vitro anti-DPV activity by 4 modes:the direct inactivity to virus, drugs’protection for cells, penetration of virus and the intracellular reproduction of virus. Use the TaqMan fluorescent quantitative PCR method to detect the copies of the virus which has reproduced, in various ways. The results showed that resveratrol plays its role by influencing the intercellular reproduction of virus.② To detect the titer changes of virus with titer method and the addition of resveratrol after the virus gets into cells for 0h,2h,4h,6h and 8h. It was found that the virus titers of medicine groups were lower than the virus infection group in each time point; Among the medicine groups, the virus titers in time point of 0-6h were obviously lower than that of 8h (p<0.01). As shown by the results, the main time period that resveratrol works against the virus lies in the former 8h after the virus gets into cells. ③ After the virus gets into cells, use TEM to reveal its reproduction process in host cells and to observe the exposure cells with the action resveratrol. According to the results, DPV’s reproduction of virus nucleic acid and the formation of its nucleocapsid in the DEFs nucleus were hindered under the influence of resveratrol. From the foregoing results, it is known that resveratrol brings its antiviral activity into play mainly within the 8h period after DPV infected the host cells. During this period, virus mainly reproduces the virus nucleic acid and expresses the relevant virus proteins in the host nucleus.5. The research on resveratrol’s inhibition of DPV immediate early phase and early phase gene transcription expression Taking β-actin as the internal reference, this research established the SYBR Green Ⅰ reverse transcription fluorescent quantitative PCR method to study resveratrol’s inhibition of gene transcriptions in immediate early and early genes of UL2, UL23, UL30, UL39, UL40, IRS and TRS. The results showed that resveratrol with various concentrations had distinct inhibition effects on the tested genes transcription. The higher the resveratrol concentration is, the lower gene transcription will be; no obvious transcription occurred in the entire tested genes within 24h when the resveratrol concentration was 31.25 μg/mL. The results revealed that resveratrol with high concentration can significantly inhibit the transcription level of genes mentioned above. Since the above-mentioned genes are the necessary genes for the reproduction of virus DNA, their transcription products involves in the reproduction process of virus nucleic acid directly; as the virus immediate early phase genes, IRS, TRS and UL23 play an important role in the transcription of early phase genes. Therefore, resveratrol’s inhibition effect on the immediate early genes could be the key reason for the inhibited DPV nucleic acid reproduction.6. The research on resveratrol in-vivo anti-DPV activity and its regulation function of the relevant cytokines To establish DPV-CHv high virulent strains cherry valley duckling duck plague model through the subcutaneous injection inoculation method. Give different doses of resveratrol on 1～7d of the inoculation by oral medication; observe for 7d after drug withdrawal. Results as following: ① All dose groups can significantly delay the onset and death time of the infected ducklings. In viral infection groups, the onset of disease started on 4d; death occurred from 5d; mortality reached 100% on 8d. In treatment groups, the onset of disease started on 5d; death occurred sporadically on 6d; mortality of each group reached 20% on 8d. A week after drug withdrawal, the mortalities of the high, medium and low groups were 80%,60% and 50%, respectively. ② Resveratrol could apparently improve the organ tissue lesion status of the infected ducklings:the systemic organs of ducklings in the viral infection groups were suffered from obvious bleeding and hyperemia, and necrotic foci were seen macroscopically in their livers; no obvious macroscopic pathological changes was found in treatment groups. In the livers, spleens and bursa of fabricius of viral infection groups, multiple necrotic foci were detected with HE staining method; in resveratrol treatment groups, no necrotic foci was detected in livers, spleens and bursa of fabricius. TEM observed that the hepatosplenic cellular structures of viral infection groups were badly damaged; nucleus and cytoplasm were filled with DPV viral particles of all growth stages. In resveratrol treatment groups, the hepatosplenic cells were with unbroken structures and no obvious visible DPV viral particles were observed in those cells. ③ Resveratrol remarkably inhibited the reproduction of virus in the infected ducklings:the rise time of viral copy number in treatment groups was 2～3d late than the viral infection groups; the rise range was obviously lower than the viral infection groups.④ With regard to the adjustment of cytokines. different doses of resveratrol has different effects on various of cytokines:when compared with viral infection groups and normal groups, it has facilitation on INF-α and INF-γ, inhibition of IL-2 and IL-12 and inconspicuous effects on IL-4; according to the comparison between different resveratrol treatment, a negative correlation was found between its concentration and its effects on INF-α, IL-2 and IL-12; high concentration refers to low content of each cytokine; the difference between INF-γ and IL-4 in different dose groups is not obvious. The mortality of high dose groups is higher than that of low dose groups, which is mainly resulted from the low cytokine content caused by high doses.In conclusion, resveratrol has good anti-DPV activity. Its anti-DPV mechanism is mainly reflected in the following two aspects:firstly, resveratrol can aim at the virus itself and effectively inhibits the transcription of DPV immediate early phase and early phase, hinders the viral nucleic acid reproduction of virus in host cells, thus to block the reproduction process of the virus to inhibit the virus; this kind of inhibition is positively related with resveratrol concentration. Secondly, by acting on animals’immunity system, resveratrol can adjust the virus-relevant cytokines to influence the organism damage of virus. This effect has complicated relationship with resveratrol doses; same dose has different influence on different cytokines. Generally, it presents a certain appearance of high dose inhibition and low dose facilitation. |
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