| Infectious Atypical Pneumonia also known as severe acute respiratory syndrome (SARS) formally declared by the World Health Organization (WHO), is a respiratory viral illness caused by a coronavirus called SARS-associated coronavirus (SARS-CoV) with the epidemiological characteristics such as high contagiousness, heavy illness, rapid progression, and causing serious harm to life. It has been recognized as a global threat to the health and economic development of life of human being.At present, a supportive therapy is the main treatment of SARS in modern medicine. Anti-virus drug and vaccine development is also concentrated by the researchers worldwide. In consideration of a long research and development cycle of a new drug and vaccine, the viral characteristic structure, its particular site of invasion and easy mutation, SARS specific treatment becomes more difficult. It is therefore globally importance to find an effective anti-coronaviral drug for the viral disease prevention and treatment.There are certain advantages for Traditional Chinese Medicine (TCM) to prevent and treat this viral disease. Based on the characteristics of SARS and his past experience of the respiratory viral infectious disease treatment, Professor Peng Sheng-quan gained the clinically notable effects of treating SARS in its early and middle stage by using Kangliyin (KLY, the compound formulation of a Chinese herbal medicine) with the functions of removing heat and damp, strengthening the stomach and removing the phlegm.As a SARS model established in a laboratory has so many safety issues to be considered. The existing safety standard in our laboratory can not allowed us to conduct a real SARS study. Infectious bronchitis virus (IBV) is the only animal infectious respiratory virus in the Coronaviridae family where SARS-CoV also belongs to. Recent researches also found that SARS-CoV and IBV had a close relationship which showed 63% homological nucleotide sequences. Even though IBV causes an acute respiratory infectious disease among chickens, it is stillrelatively safe to the research staff and environment under strict experimental conditions. Therefore, it is our consideration of choosing IBV as a SARS-CoV substitute to build a respiratory coronavirus infection like model.Based on what have been mentioned above, the main aims of this research are to establish the substituted respiratory coronavirus infection model;and by using this model to evaluate the pharmacological action of KLY and to some extent understand its mechanism of treating respiratory coronavirus infection. As a result, a further KLY use in treating respiratory coronavirus infection may be developed.Section I An in vitro study of respiratory virus infection treated with KLY.Objective: To explore the effects of KLY in treating three strains of respiratory virus infection in vitro, i.e. RSV (Respiratory Syncytial Virus), Ad3 (Adenovirus) and FM1 (Influenza Type A virus).Methods: Toxicity tests were performed on MRC-5 cells and MDCK cells treated with KLY in the cell culture media. These were followed by the measurement of anti-RSV's and anti-Ad3's activity of KLY in MRC-5 cells, and the anti-FM1's activity measurement of KLY in MDCK cells.Results: The TC50 (50% toxicity concentration) of KLY on MRC-5 cells and MDCK cells were 7.62 mg/ml and 21.33 mg/ml respectively, and the TCO (none toxicity concentration) were 5.50mg/ml and 9.38mg/ml respectively after adding KLY in the cell culture media. The IC50 (50% inhibition concentration) of KLY on RSV, Ad3 and FM1 were >7.62mg/ml, >7.62mg/ml, and >22 mg/ml respectively when treated with KLY in vitro. The Tl (treatment index, Tl= TC50/ IC50) on RSV, Ad3 and FM1 were all <1 when treated by KLY in vitro.Conclusion: KLY had more toxicity on MRC-5 and MDCK cells in the cell culture media, and had no significant effects on RSV, Ad3 and FM1 in vitro.Section II The effects of Kangliyin on the Respiratory Coronavirus Infection ModelPart I Respiratory coronavirus IBV propagation and identification Objective: To establish reliable methods of IBV identification and diagnosis. Methods: 1. IBV M41 strain was inoculated in the allantoic cavity of 9-day-old to 10-day-old eggs. The eggs were incubated at 37°C for 48h. The allantoic fluids were harvested from the eggs. 2. IBV M41 strain was initially assayed by using the experiments involving dwarfing of the chicken embryo and interference of NDV-B1 propagation. 3. According to the whole gene sequence of IBV-M41 registered in GeneBank, a pair of primers spanning N gene sequence and 3' end of genomic non-coding region sequence of IBV was designed to test IBV-M41 in the allantoic fluids by using reverse transcriptase polymerase chain reaction (RT-PCR). Results: The results of the experiments involving dwarfing of the chicken embryo and interference of NDV-B1 propagation were positive, and initially manifested the existence of IBV in the allantoic fluids. A line of special strip was amplifiedsuccessfully in the allantoic fluids by using RT-PCR, and the amplified products had 1404bp which were in line with its design.Conclusion: The experiments involving dwarfing of the chicken embryo and interference of NDV-B1 propagation provided the useful data for IBV identification and diagnosis. The primer designed in accordance with IBV gene sequence has a higher specificity to IBV, and the method of RT-PCR is an accurate and rapid measurement to identify IBV.Part II Studies on Respiratory Coronavirus IBV Infected Chick Model Objective: To establish a reliable IBV infected chick disease model. Methods: 7-day-old chicks were inoculated with respiration type of IBV M41 strain by the nasal -ocular route. An identical volume of normal saline was inoculated as controls. All the chicks were breed in a low temperature and highly moisturized controlled environment. General and respiratory symptoms, body weight and anal temperature were observed. Chicks with sinificant symptoms would be anatomized, and the pathological changes of bronchus and lung tissue were observed. Finally, according to the whole gene sequence of IBV-M41 registered in GeneBank, a pair of primers spanning N gene sequence and 3' end of genomic non-coding region sequence of IBV was designed to test IBV-M41 in the lung and bronchus tissues by using the method of reverse transcriptase polymerase chain reaction (RT-PCR). Results: Under the physical environment of low temperature and high moisturization, the IBV infected chicks appeared with clinical symptoms and signs of high fever, sneezing, coughing, tracheal rale, nasal discharge, labored breathing, inappetence, diarrhea, cachexia, and narcoma. The pathological appearances of the models showed typical bronchitis of lung and bronchus tissues. A line of special strip was amplified successfully in the infected tissues by using RT-PCR, and the amplified products had 1404bp which were in line with its design. Conclusion: Respiratory IBV infected disease model could be replicated successfully by inoculating the respiration type of IBV M41 standard strain and under the physical environment of low temperature and high moisturization. This model also showed with the TCM syndrome of damp-heat retardarce and dysfunction of lung caused by respiratory virus infection.Part III Experimental Studies of the effects of Kangliyin on Respiratory Coronavirus Infection ModelObjective: To study the effects of KLY treatment on the IBV infection models and to evaluate its effects on the IBV N gene expression and the IBV antibody generation. Methods: 7-day-old chicks were inoculated with the respiration type of IBV M41 strain by the nasal -ocular route. They were divided into five groups by stratified random sampling, which included model group, three treatment groups(known as low-dosage group, middle-dosage group and high-dosage group), and positive control group(ganciclovir group). The chicks were also inoculated with normal saline as the normal control group. All the chicks were bred in a low temperature and highly moisturized controlled environment. The model group and normal controlgroup were fed with sterile normal saline, while the three treatment groups were irrigated with KLY compound at the dosages of 1Og/kg.d, 20g/kg.d and 40g/kg.d respectively. Positive control group were irrigated with ganciclovir at the dosage of 0.004g/kg.d. These feeds had been lasted for 5 to 7 days. (1) The clinical symptoms, body temperature and mortality rate of these IBV infected models were observed. (2) According to the whole nucleotide gene and N gene sequence of IBV-M41 and the nucleotide sequence of the chick cytoplasmic 3-actin gene registered in GeneBank, two pairs of primers were designed in real time PCR. The relative quantities of IBV-M41 in the lung and bronchus tissues were assayed by using real-time florescent quantitative polymerase chain reaction (Real-time PCR) at the end of the experiment;and the pathological changes of all groups' bronchus and lung tissues were examined. (3) The levels of IBV antibody in the serum of each group were also checked by using ELISA method at the end of the study.Results: (1) Compared with the model group, KLY compound could significantly lower the high body temperature, improve the general clinical symptoms and respiratory symptoms, and decrease the mortality rate of these IBV infected models in the three treatment groups. (2) Compared with the model group, KLY compound could significantly inhibit the IBV N gene's expression in the three treatment groups, and could improve the pathological changes of the bronchus and lung tissues of the IBV infected model. (3) Compared with the normal control group, the serum level of IBV antibody in the model group was significantly increased. But the result was still negative. Compared with the model group, KLY compound could significantly increase the serum titer of IBV antibody in the three treatment groups. Both middle-dosage and high-dosage groups were positive while the low-dosage group remained negative.Conclusion: KLY could inhibit the viral replication, improve the general clinical symptoms and respiratory symptoms, lower the high body temperature, improve the TCM clinical symptoms in the IBV infected model, and decrease the mortality rate of these IBV models. KLY could repair the pathological damage in the bronchus and lung tissues of the IBV model, and increase the serum titer of the antibody against IBV which would eventually speed up the chick's recovery from IBV infection. This may be one of reasons that KLY is effective in treating the IBV infected model.There are certain peculiarities and new ideas in this study. Firstly, it was the model established by coronavirus infection in vivo. This model has not been mentioned in the pharmacological literatures of the anti-virus drugs promulgated by Ministry of Health, Peoples Republic of China before. This respiratory coronavirus infected model which was established by choosing IBV as a substitute of SARS-CoV would fill up the blank in this area. It would provide another channel for screening an effective Chinese or Western medical drug. To a certain extent, this model also matched the TCM syndrome of respiratory virus infection. Therefore it had an important value for evaluating an anti-viral drug, especially a Chinese medicine compound with the functions of removing heat and damp, strengthening the stomach and removing the phlegm. Secondly, although the respiratory coronavirusinfection model was established by a none SARS-CoV virus infection in this study, there was still a co-relation between the two types of virus. This model have the following advantages over the others: easy establishment, good reproducibility, safety and no special laboratory need, short experimental period, economy, and limit threat of causing biological disaster, ease of widely use, and several models establishment at one time. All these will be propitious for studying the anti-coronaviral drugs, especially for an initial screening of an anti-coronaviral drug. Finally, a new assay technique was adopted known as real-time florescent quantitative polymerase chain reaction (Real-time PCR) in the study of IBV models. This newly developed technique is a test of accurately quantitative measurement of gene in recent year. It has the features of high sensitivity, specificity, reliability, and reproducibility, multi-reaction, high automatization, very little pollution and real-time etc.. This real-time PCR assay used in this study provides an accurate quantitative measurement to the virus in tissues which brings the results with more accuracy and reliabilityo...
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