| Because of influenza A virusvariation fast, anti-virus chemical drug having great side effect, easy producing drug resistance, and preparation of effective vaccines very difficult, so looking for a safe, convenient and effective prevention and control drugs of influenza A virus become a challenging research topic at present.Egg yolk immunoglobulin (IgY) has many advantages. It provides an inexpensive, effective and stablesource of antibodies for the passive immunization of animalssufferingfrom different kinds of diseases. In the aspact of prevention, diagnosis and treatment of viral diseases, IgY has wide application prospects.In this study, a specific IgY was produced in hens immunizedwith inactivated-influenza A virus. The antiviral activities of the specific IgYin vitroandin vivo were investigated. The detection of influenza A virus by use of IgY was carried out as well. It could provide a theoretical basis for the application of the specificIgY in theprevention, treatment and testing of influenza A virus.This study mainly includes four parts:Part 1:Preparation of Egg yolk immunoglobulin against influenza A virus1.1 The specificIgY was produced in hens respectively immunized with inactivated influenza A virus including A/Guangdong/732/2009(H1N1)pdm09, A/ California /7/2009 (H1N1)pdm09, A/Guangdong/622/2009 (H1N1) and A/Guangdong/749/2009 (H3N2). After PEG precipitation, purity of the antibody was 95.80% and recovery rate reached to 93.01%.1.2 By ELISA test, antibodies from different antigens had consistent trend of titers. The highest titer of 1.0 mg/mL IgY reached 3200, which could be sustained for over 6 weeksin the egg yolk. High titer antibodies (>2500) could be kept for over 10 weeks in the egg yolk.Part 2 In vitroactivity evaluation of IgY against influenza A H1N1 influenza virusThe antiviral activity of IgY against A (H1N1) influenza virus A/Guangdong/ 732/2009 (H1N1) pdm09 was evaluated at the molecular level and cellular level.2.1 The study of AGAR diffusion and fluorescence immune response showed that IgY could combine with and precipitate virus particles. The study of Western Blot and HI test clarified that IgY could be specifically bound with major membrane proteins including neuraminidase (NA), hemagglutinin (HA), nucleoprotein (NP) and matrix proteins (M) on the surface of the virus. And it could effectively suppress erythrocyte aggregation caused by the virus. The maximum dilution multiple of 1.0 mg/mL IgY that could completely inhibit erythrocyte agglutination was 128.2.2 Plaque reduction test indicated that IgY could effectively neutralize the virus. IgY could directly inhibit virus adsorption of the host cell, IC50<2.0 μg/mL. It also revealed that IgY could effectively prevent virus from attaching to cells and could inhibit replication and proliferation of virus in cells. IgY has significant and stable antiviral activity in vitro.Part 3 Antiviral Activity of anti-A(H1N1) IgY was studied in mice (in vivo)3.1 Effect of IgY on the survival rate of mice infected by A/Guangdong/732/ 2009 (H1N1)pdm09The BABL/C mice were randomly divided into fifteen groups:normal group, model group, positive drug-treated group, specific IgY-treated groups (Prevention groups, Therapy groups and Prevention-treatment groups. There were 3 different concentrations in each group.), negative IgY groups (3 different concentrations). Then, exceptnormal group, BABL/C mice were inoculated intranasally with a lethal dose of 5×LD50 of A/Guangdong/732/2009 (H1N1)pdm09 virus resuspended in 50.0 μL PBS per animal in the rest of groups. Treatment was provided only once in 2 hours before intranasal infection in Prevention groups. In Therapy groups, treatment was provided once a day for 4 days and the first time of treatment was at 2 hours after intranasal infection. In Prevention-treatment groups, treatment was provided before and after infection just like Prevention groups and Treatment groups. The effect of IgY on the life and weight protection of infected mice after 14 days of inoeulation was observed. Results are as follows:① The survival rate of model group and negative IgY groups was 10%. The survival rate of specific IgY groups was 90% or above compared with the model group P<0.001.② The weight of mice in model group and negative IgY groups decreased at first and then picked up in a upward trend, but was significantly lower than that in the normal group. The weight of mice in specific IgY groups was significantly higher than that in model group. The changes of body weight and survival rate were the same in medium-dose group and positive drug-treated group.3.2 Effect of IgY on the lung injury and virus propagation of mice infected by A/Guangdong/732/2009 (H1N1)pdm09The BABL/C mice were randomly divided into six groups:normal group, model group, positive drug-treated group, specific IgY-treated groups (Prevention group, Therapy group and Prevention-treatment group). The methods of infection and administration were the same with the methods in 3.1. On the third day and sixth day after virus infection, tested mouse were sacrificed and the lung indexes were analyzed. Lung tissue with viral load was tested by RT-PCR. The virus titer of lung tissue was tested by HA. Routine HE stained sections were examined. The results are as follows:① Based on the observation of animal subjects at different time Points, the model group presented a gradual upward trend of the lung index, while the lung indexes of specific IgY groups were significantly lower than that of the model group. Actually, the lung indexes of specific IgY groups were quite different, on the third day after infection, p<0.05, and on the sixth day after infection, p< 0.01.② Based on the observation of animal subjects at different time Points, the lung tissue viral load and lung virus titer in the model group were much higher than those in specific IgY groups. The lung tissue viral load and lung virus titer of Prevention-treatment in specific IgY groups were extremely different, p< 0.001; all other groups treated or prevented by specific IgY had a great difference, p<0.01.③ HE staining under light micro scope was applied to observe the specific IgY groups mice. The result was that the basic structure of the lung tissue was intact and infiltration and necrosis of the lung tissue were both in a low level.Part 4 Establishment and application of an immune analytical method with Surface enhanced Raman spectroscopy marker to detect influenza A (H1N1) virusGold sol was successfully prepared with sodium citrate as reducing agent. 4,4’-Bipyridine as biomarkers for Raman preparation was applied to mark gold sol. Immunegold sol was then preparedby combining IgY with the tagged gold sol. The spectrum of the marked immune gold sol was consistent with the markers. The solid-phasedIgY antibody was assembled in hollow glass slide with the method of silane and aldehyde.With optimized experimental steps, the immune analytical method of detecting influenza A (H1N1) virus with the surface enhanced Raman spectroscopy marker was established. In the test of influenza A (H1N1) virus, the virus titer was in the range of 0.2 TCID50 to 2000 TCID50; the 4,4’-Bipyridine SERS signal was linear to antigen concentration; the linear equation was Y=2348.4X-1525.8 and the correlation coefficient was 0.9998. |
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