Research On Genetic Identification And Anti-viral Effect Of Edible Bird's Nest

Objectives:The edible bird's nest (EBN) mainly comprises a secretion of the salivary gland of several species of Aerodramus or Collocallia genus in the Apodidae family found predominately in Southeast Asia. EBN is a precious functional food and therapeutic herbal medicine that has been used for several hundred years in China. Due to the increasing price of genuine EBN incurred by the limited supply, adulteration and substitution are often found in the EBN commercial market. Furthermore, it is traditionally considered that the market value of EBN is closely related to the species and habitate of swiftlets producing EBN. However, the currently available authentication methods for EBN, such as ultra violet spectrophotometry, infrared spectrometry, gas chromatography, protein electrophoresis and assay of chemical component which could indicate the authentication and purity of EBN by detecting the differences in chemical components between EBN and its counterfeits, cannot identify the genetic source and habitate of EBN. In recent years, molecular biology provides us a powerful tool to genetically identify species from samples with DNA. Therefore, identification of the genetic source of EBN would bring breakthrough in the field of EBN quality assurance and would provide importance complement to the current analysis method of EBN. In addition, the efficacy as well as the efficacy-value relation of EBN has been in doubt in academia all along. In response to these issues, this paper discusses the application of DNA sequencing and analysis technique in the genetic identification of EBN with an expectation to evaluate the EBN quality. In the mean time, the effect of anti-influenza virus and the mechanism of EBN are studied with a view to develop a model for evaluation of the EBN efficacy and conduct a pilot study of the quality-efficacy relation of EBN.Methods and Results:1. Genetic identification of EBNThe research work of genetic identification of EBN by using DNA sequencing and analysis technique mainly include exaction of DNA with satisfactory quality, amplification and sequencing of target gene, identification the genetic species by sequence analysis.In extraction of DNA, this paper investigated the performances of four DNA extracting method, i.e. kit method, alkaline lysis method, phenol method, high-salt low-pH method. It was revealed that DNA could be obtained by phenol method. The phenol method was further optimized in the aspects of digest buffer formula, purification method and precipitation conditions. The DNA extracting method was finalized as follows:the EBN protein was dissolved and the cells integrating with saliva was digested by SDS digestion buffer with high concentration of NaCl, the dissolved protein was gradually precipitated in high NaCl concentration environment so that it could be removed by centrifugalization, after that, the DNA solution was extracted with choloroform/isoamyl alcohol mixture to denature and remove the residue protein, and then CTAB was added to further precipitate glycoprotein as CTAB could form soluble complex with DNA but insoluble complex with polysaccharide in high NaCl concentration environment, the solution was again extracted with choloroform/isoamyl alcohol mixture to remove glycoprotein precipitation, finally, DNA was precipitated by pre-cooled isoamyl alcohol in room temperature for a short time to minimize the co-precipitation with glycoprotein. By using this established method, DNA in the EBN was obtained with satisfactory quality for PCR amplification.Before DNA ampliation, the target gene for research should be determined. After a search on Genbank, a total of 82 sequences of Cytb gene (whole or partial) from twenty species from Aerodramus genus and ten species from Collocalia genus have been identified, indicating that the number of sequences of Cytb gene are the largest among other genes from swiftlets. Cytb gene has been widely used in the study of genetic differences among species of bird due to its faster evolutionary rate. Therefore, Cytb gene was targeted for sequence analysis with an expectation to reveal the gene differences between swiftlets from different species or habit so as to identify the EBN genetic source.The obtained DNA in trace amount should be amplified before sequencing. However, the success rate and sensibility of PCR for DNA obtained from EBN was low because of the low abundance of DNA and the interference by glycoprotein. We then investigated methods for improving the PCR success rate and sensibility, including increasing the concentration of Mg2+ in PCR buffer, PCR with enhancer, booster PCR, conducting PCR for the second time, PCR in small volumn buffer, PCR with decreased anneal temperature and nest PCR. Among these method, nest PCR was found to be the best choice for this work. Nest PCR could also help to extricate from the exogenous DNA contamination by improving the specificity of PCR as two pairs of PCR primers are used to amplify a fragment of DNA. The established nest PCR procedure are as follows:the complete Cytb was firstly amplifyied with primers ND5 and Thr, the amplifyication product was then used as a template for three different second PCRs using three different pairs of primers to amplify the left, middle and right segment of Cytb. The three segments of Cytb obtained by nest PCR were sequenced and compared in terms of their sequencing signal, PCR success rate and the percentages of informative sites to select the segment suitable for constructing phylogenetic trees. The results showed that the middle segment had the clearest sequencing signals, highest PCR success rate and highest percentage of parsimony sites. Therefore, the middle segment was selected for further analysis for genetic identification of EBN.Sequence analysis of target gene was performed by constructing phytogenetic tree. Firstly, the sample sequences obtained were imported into nucleotide BLAST for retrieving similar sequences from GenBank and the similarity between the sample sequences and the first hit sequence (the similarest sequence) from GenBank was calculated to preliminary identify the genetic source of EBN sample. Secondly, phylogenetic trees were constructed with all sample sequences and sequences retrieved from GenBank together with Amazilia tzacatl sequence as an outgroup sequence. As BLAST search results showed that the top 100 hit sequences were from Aerodramus or Apus genus, all cytb sequences of species from these two genus in GenBank have been collected for phylogenetic tree construction. To verify the reliability of the results, three phylogenetic trees were constructed using three different calculation methods, i.e. using Neighbor Joining (NJ) method to construct NJ tree, using Maximum Parsimony (MP) method to construct MP and using Mrbayes 3.12 to construct Bayesian tree. Fourteen samples were involved in tree construction. They were 11 samples of Indonisian EBN,1 sample of a swiftlet found roosting in the cave having EBN in Indonesia,1 sample of ready-to-eat EBN soup product claimed to be produced by Indonesia EBN and 1 sample of Huaiji EBN obtained from a cave(Yan cave) in Huanji country having swiftlet nests. The results showed that the three tree shared similar topology with all sequences separated into two groups:Aerodramus or Apus. The EBN samples and swiftlet sample were all aligned with Aerodramus group and clustered with Aerodramus fuciphaga, suggesting that Aerodramus fuciphaga was the genetic source of them. As for the sequence from Huaiji EBN, it was aligned with the Apus group and clustered with Apus affinis and Apus nipalensis equally in the NJ tree and Bayesian tree, but only with Apus nipalensis in the MP tree. Apus affinis and Apus nipalensis were close in genetic relationship and the latter one is treated as a subspecies of the former one in East Asia which was named Apus affinis subfurcatus. Therefore, it was concluded that Apus nipalensis was the genetic origin of the sample of Huaiji EBN according to phylogenetic tree analysis combined with habitat analysis.2. Research on antiviral effect EBNAccording to traditional Chinese medicine theory, EBN has effects of moisterning lung and nourishing yin, transforming phlegm and suppressing cough, supplementing the center and boosting qi. Pharmaceutical effects of EBN included antivirus, potentiating the mitogenic response of cells and improving immunologic function. The indication of EBN in respiratory and gastrointestinal diseases may be contributed by the antiviral effect of EBN which is related to the silaoprotein. Therefore, research on the EBN antiviral effect is essential for clarification of the efficacy mechanism, product development and substitute research. This paper then selected the silaoprotein-based antiviral effect for efficacy study of EBN. The preliminary study was conducted using in vitro antivirus model. The EBN is usually oral ingested after stewed, which makes the EBN protein degraded when contact acid, base and enzyme under gastrointestinal tract so that the activities of EBN may be affected. Taking this into account, this study was divided into two parts, the first part is to prepare sample and the second part is to conduct preliminary study on in vitro antiviral effects.Sample preparation should simulate the disposal process of EBN undergone when steaming or entering gastrointestinal tract. Therefore, three different process method were applied for antiviral study:1. EBN water extract (EWE):extracting at 80℃for 1h; 2. Simulated gastric fluid digestion contents of EBN water extract (SGF-EWE):added an equal volume of 2xSGF to the EWE and heating at 37℃for 10 min.3. Simulated intestinal fluid digestion contents of EBN water extract (SIF-EWE):added an equal volume of 2xSIF to the EWE and heating at 37℃for 10 min. SDS polyacrylamide gel electrophoresis was conducted to detect the degradation degree of these 3 samples. Results showed that the protein molecular of EWE, which did not undergo digestion by SGF or SIF, was mostly near 170 kDa. After digestion with SIF or SGF, the protein was degraded in 1 min and the degradation products were still high molecular protein, about 55～100 KDa. The results indicated that the chemical component of EBN would changed in gastrointestinal tract.In vitro antivirus model was used in the preliminary study. The effects of the 3 prepared samples on the activity of H5N1 avian influenza false virus and VSV-G false virus, the coagulation effect of H5, H7 and H9 type antigen, as well as the activity of N1 neuramidinase. Results showed that all the 3 prepared samples had dose-dependent inhibitory effect on the activity of H5N1 avian influenza false. The EWE-SIF beared the strongest inhibitory effect with the median effective concentration at 2.30±0.62, the EWE-SGF followed with the median effective concentration at 4.00±3.60, and the EWE had the largest median effective concentration at 5.29±1.79. In order to further investigate the mechanism of the inhibitory effect of EBN on H5N1 avian influenza false virus, studies on the effect of the 3 prepared samples on VSV-G false virus were conducted with the results being all negative. The presence of envelope protein in H5N1 avian influenza false virus is the only one difference between H5N1 avian influenza false virus and VSV-G false virus. The results that the prepared samples showing inhibitory effect on the former but not on the latter indicated the action target was the envelope protein of H5N1 avian influenza false virus. There are two importance protein related to the infection activity of the H5N1 avian influenza false virus on the envelope protein, i.e. hemagglutinin (HA) and N1 type neuramindase (NA). In order to identify which is the function protein, this study investigated the influence on the coagulation effect of H5, H7 and H9 type antigen and the direct inhibitory effect on NA. Results showed that the 3 prepared samples have inhibitory effect on the coagulation effect of H5, H7 and H9 type antigen but not on NA, indicating the antiviral effect of EBN may be contributed to the inhibitory effect on coagulation effect of the HA on envelope protein. In another words, HA was the action target.Conclusion:The paper describes, for the first time, a method for genetic identification of EBN and its product such as instant EBN soup based on DNA sequencing technique and phylogenetic analysis. Challenges in genetic identification of EBN using DNA analysis technique are poorness of DNA in EBN, interference of glycoprotein and exogenous DNA contamination. These Challenges have been overcome after optimization of DNA extraction method to get rid of the interference of glycoprotein, as well as improvement of PCR sensitivity and specitifity by using nest PCR technique. Results achieved from this method are stable and repeatable. The DNA extraction and PCR amplification method in this study is simple in operation, inexpensive, no need of special reagents and equipment. With the increasing popularization of DNA sequencing technology, this method will be widely used. On the aspect of EBN therapeutic effect, this paper conducted research on the antiviral effect of EBN. It is demonstrated, for the first time, that the EBN has antiviral effect on H5N1 avian influenza false virus with the HA on the envelope protein being the action target. Previous research showed that EBNs from different production environment (cave EBN and house EBN) showed different potence of antiviral effect. The antiviral effect of cave EBN was much stronger than that of house EBN. The peak of O-acetyl sialic acid in house EBN was much higher than that in cave EBN in Fluorometric HPLC. Therefore, it could be assumed that the different potence of antiviral effect between house EBN and cave ENB may be associated with the different structure of sialic acid which could affect the binding activity of EBN to virus, indicating the importance of the identification of EBN genetic source. Therefore, we could evaluate the therapeutic effect of EBN through the antiviral effect of EBN and evaluate the cost-effect relation of EBN together with DNA identification technology.