Neuraminidase Inhibitors Screening From TCMs And Their Anti-influenza A Virus Effects

Epidemic influenza is an acute viral respiratory tract infectious disease caused by Influenza virus that can severely impair human health. Influenza remains a serious health problem worldwide, causing the deaths of millions of people and imposing substantial economic costs every year. In the 20th century, five influenza pandemics emerged and killed tens of millions of people. The 2009 outbreak of swine origin H1N1 virus rapidly transmitted and spread among humans. Strategies for dealing with influenza are based on annual immunization and antiviral drugs. For that reason, anti-influenza drugs are crucial for the control of influenza, and in the face of a pandemic virus they would be the most important short-term resource. Information about the optimal use of the currently available anti-influenza drugs is needed.Influenza is caused by influenza virus and the neuraminidase(NA)encoded by the virus has vital role in the life cycle of the virus for its recognition, entry and release from the cells they infect. In the mean time, the active site of NA is antigenically conserved among all clinically strains. As a result, NA inhibitor have been developed and used as an effective and promising influenza drugs. Zanamivir and Oseltamivir design have been used in clinics. However, numerous reports have been seen with drug resistance to these NA inhibitors, and these resistances are mostly possibly caused by the mutations in the NA of the virus. To overcome the drug resistance, novel NA inhibitors with new structure scaffold are needed.Influenza falls into the category of exopathic disease in Traditonal Chinese Medicine(TCM), TCMs have been used to prevent and treat influenza with a long history,now which is believed to be an important research way for the prevention and treatment of influenza. However, only in recent years researchers have started to test these TCMs experimentally in cell culture and in animal models. A variety of polyphenols, flavonoids, saponins, glucosidesand alkaloids isolated from medicinal herbs have been studied extensively and tested for anti-influenza activity, as reflected by their ability to block adherence, penetration, duplication and/or maturation during the course of viral propagation. The molecular mechanism of these compounds in viral inhibition started to unfold in recent years.Even several reports showed that TCMs could be used to suppress viral infection, little work has been done to test whether these TCMs could inhibit NA activities. Thus it was of great interest to investigate NA inhibitory effect in the widely used TCMs and test their ability to suppress viral infection.In this study, we tested the inhibitory activity of water extracts from TCMs towards NA. Next, we evaluated the anti-influenza-virus effect of these TCMs water extacts, which exhibited strong inhibitory activities to NA, experimentally in cell culture and in animal models. The result will help us to elucidate the new mechanism of the anti-influenza-virus therapeutic effect of these TCMs and give new insight in the field of NA inhibitor development.First, we tested water extracts of 439 TCMs for their inhibitory activities towards NA. The TCMs were separately decocted, lyophilized and prepared to a final concentraion of 10mg/mL TCM water extracts, respectively. We used the classic MUNANA-based NA activity assay for screening the NAI from TCMs, Both NA in the allantoic fluid of H1N1 virus infected eggs and from C. perfringens were used in the assay. Here we tested the inhibitory activity of water extracts from 439 (TCMs) towards NA. Five TCMs, Duchesnea indica, Liquidambar formosana, Lithospermum erythrorhizo, Melia toosendan, and Prunella vulgaris, exerted potent inhibitory activity towards NA.Next, we tested the viral inhibitory activity of these NA inhibiting TCMs in H1N1 viral infected MDCK cells. In the MDCK cell culture experiment in vitro, the cytotoxic effect of different concentrations of the five TCMs was studied to determine the suitable non-toxic concentration. It is useful to choose the appropriate concentration range for the next experiment in influenza virus infection. Although higher concentration of the TCMs caused a reduction in the cell viability, the 5 TCMs reduced CPE in MDCK cells and also reduced the virus load in a dose-dependent manner.The cellular anti-viral effect was further supported by the animal model test. Melia toosendan was randomly selected from the 5 TCMs that showed NA inhibitory activity and viral protection and used to treat the infected mice.We established the mouse model of influenza pneumonia. FM1 virus was used to establish a mouse model. After being inoculated intranasally with 10 LD₅₀ of FM1 virus, the mice were observed every day for recording the morbidity and the mortality. On day 1 or 2 post infection, most of the infected mice began to exhibit clinical signs of influenza, including ruffle fur, hunched posture, weight loss. To monitor the viral infection in lung, the mice were sacrificed at day 0, 3, 5, 7 post infection and their lungs were removed for testing the lung index and preparing homogenates that were used to test the FM1 virus load by NA activity assay, The results showed that the lung index and the viral load in lung homogenate in mice began to increase on day 3, on day 7 post infection, the lung index and virus load in the lung homogenate reached to the maximum. Within ten days, all the infected mice succumbed, indicating that the strong inflammatory reaction was associated with the death.Upon the results, the mouse infected with FM1 virus was adopted as an animal model to evaluate the effects of Melia toosendan on the influenza pneumonia caused by FM1. We studied the effect of Melia toosendan on the morbidity, mortality, weight loss and mean day to death (MDD) caused by influenza virus. On day 0, mice were inoculated intranasally with 10 LD₅₀ of FM1 virus (n=10). Melia toosendan at a dose of 40, 80, or 160 mg/kg of body weight or Tamiflu at a dose of 50 mg/kg of body weight was gavaged to the mice once daily for 15 days, with the first dose administered 2 day before infection., Normal and PBS-treated virus control mice were included in the same treatment schedule, and then observed and weighted daily. Mice infected with virus without treatment that had survived exhibited an obvious weight loss. When treated with Melia toosendan at a dose of 40 mg/kg, similar weight loss was observed. However, when the animals were treated with 80 mg/kg Melia toosendan significant regain of the weight starting on day 12 was observed, despite the similar initial weight loss in the first 10 days. Remarkably, group that treated with 160 mg/kg Melia toosendan showed mini weight loss through out the infection and treatment, these animals kept relatively steady weight and was comparable with the un-infected and Tamiflu-treated ones. To record the mortality, the mice were observed for 15 days after infected by virus. On day 6 post infection, the mice in three groups began to die. On day 15, the mortality rates of the mice treated with PBS successively increased to 90%, whereas the mortality rates of the mice treated with 40mg/kg, 80mg/kg or 160mg/kg Melia toosendan decreased to 80%, 60% or 50%, respectively. As expressed by mean survival, the mice treated with 160mg/kg, 80 mg/kg, 40mg/kg Melia toosendan or PBS survived for 11.9±2.4, 11.0±2.6, 9.5±4.4 or 7.4±3.0 days, respectively, indicating the more potential of Melia toosendan on inhibiting lung injury .At the end of experiments, their lungs of the mice were removed for gross observation and histopathological examination. The lungs of the mice treated with PBS displayed edema, consolidation and profuse hemorrhage. Unexpectedly, Melia toosendan could almost completely prohibit the development of the changes. Histopathologically, mice treated with PBS showed thickened and congested alveolar walls, intra-alveolar edema, and numerous infiltrated neutrophils in their lung tissues. In contrast, mice treated with Melia toosendan showed no or much less the changes in their lung tissues. the pathological score of the lung tissue of Melia toosendan-treated mice was lower than that in the mice treated with PBS group. The results indicated that Melia toosendan could lessen the of mice infected with influenza virus.Based on the weights of the lungs of the sacrificed mice, lung index was determined. The lung index of the mice infected with FM1 virus increased significantly, compared to that in normal mice, indicating that a strong inflammatory reaction was induced by FM1 virus in lung of the model mice. The lung index of the mice in Melia toosendan group was lower than that in the PBS group, implying that Melia toosendan could significantly inhibit the FM1 virus-induced lung injury.The viral load in mouse lung homogenate was measured by NA activity assay. The viral load of mouse lung in Melia toosendan group was lower than that in the PBS group, implying that Melia toosendan could inhibit FM1 virus replication in lung tissue of mice.Although we have shown that extracts from the above mentioned TCMs possessed inhibitory activities towards NA, the active component in these herbs is still waiting to be identified. Further purification and identification of the active compound may help us to elucidate the mechanism of the therapeutic effect of these TCMs and give new insight in the field of NA inhibitor development...