Significance of the study
According to the data of World Health Organization (2018), more than 3 million people lose their life due to respiratory tract diseases worldwide. there are numerous kind of virus and bacteria has been identified that causes disease related to lower and upper respiratory tract such as respiratory syncytial virus, influenza virus and parainfluenza virus, that are estimated to cause more than 80 percent of the deaths due to respiratory tract infections (WHO). However, recently in the western region of the United States of America, respiratory disease resulting in pneumonia and lymph-proliferation has become prevalent and according to some primary researches, the infection is caused by an unknown Coronavirus, that affects wild rodents found in deserts and infects their epithelial cells and T-Lymphocytes (Lysholm). The primary aim of this assignment is to understand the nature of the pathogen and characterize it further determine the structural component that will be beneficial in production of a specific vaccine.
Innovation (Literature review)
After detection of the virus, the prime research question is about the structural component which is useful in the production of vaccine against corona virus. This is important as creating vaccine that has low response against the target virus cannot reduce the occurrence of disease. In a research article by Weaver et al. (pp. 1087-1097), the research was conducted to understand and characterize the topology of Chikungunya virus and develop vaccine against it. Further the researcher determined the potential markers in the chikungunya virus and decided that the outer membrane will be useful for the vaccine preparation as the outer membrane protein has the ability to increase the immune response after injection that can help to generate a load of antibodies in the blood serum of immune people. However, the another researchers Haynes et al. (pp. 422-423), were failed to immune people against HIV, using the HIV envelop vaccine 1, which was prepared by creating monoclonal antibodies that is envelop specific virus against the HIV-1 infection. However, maximum of the researchers such as Liang, Zhao et al. has identified virus envelop vaccines for the vaccine preparation as it eliminates the re-occurrence of infection as well as helps to generate higher antibody pool within the human body to make it immune against the virus (Song, Daesub and Bongkyun, pp. 167-170).
Plan of the study
The two primary aims that will be investigated during the study are
- Identification of the virus that is infecting humans in western region of USA and raise specific antibodies against that.
- Conduct literature review to finalize the structural component pf virus that will be utilized in vaccine preparation.
The test hypothesis will be creating vaccine using viral envelop antigen so that safe vaccine can be produced.
H1: the viral envelop will help to create vaccine against corona virus
H0: the viral envelop used vaccine will not be able to stop the disease from spreading.
The testable prediction of the process will be either generation of an effective and useful vaccine against the coronavirus or an ineffective virus that is unable to increase the antibody loads within the body.
Importance of the vaccine
The Coronavirus is known to infect canine and wild rodents and affecting their t-lymphocytes and epithelial cells, however, the recent incident of western region of USA and the outbreak of human coronavirus made the preparation of vaccine important. Despite of the emerged problem, no FDA approved drug or vaccination is present against this viral disorder (Momattin et al., pp. 792-798). However, according to the Governmental website of center for disease control and prevention, there are two kind of human corona virus that are known to infect humans such as the Middle East Respiratory Syndrome Coronavirus or (MERS-CoV) and the SARS-CoV. The MERS-CoV has been reported in only two patients in the USA. This fact implements that in near future there is a possibility of outbreak of MERS-CoV in USA. Therefore the vaccine preparation is important (Lu et al., pp. 468-478).
The virus causing respiratory infection in humans are known as the human corona virus and for the detection of the virus, several molecular biology techniques can be used such as monoclonal and polyclonal antibodies, immunoprecipitation based techniques, solid phase antibody assay, flow cytometer and direct viral detection methods. For the detection of this virus, the monoclonal and polyclonal antibody will be used (Lysholm et al., pp. ).
Polyclonal and monoclonal antibody
The polyclonal and monoclonal antibodies are produced in live lab animals or in tissue cultures. A lab animal will be infected with the coronavirus repeatedly. Further for the polyclonal antibody, the activated B cells will produce B plasma cells and memory B cells (Wine et al., pp. 2993-2995). The later will possess polyclonal antibodies and after the collection of antiserum from the animal will help to attain polyclonal antibody. However, for the monoclonal or more specific detection, the spleen cells will be collected from the animal and after combining it with immortal myeloma cells and proliferation, specific antibody against the desired structure of the virus can be produced (Qiu et al., pp. 1-10). It should be mentioned here that for the vaccine development, the viral outer membrane will be targeted as heat denatured or deactivated RNA vaccines has the tendency to revert to its virulent nature within the human body (Wine et al., pp. 2995-2998). Furthermore, after the completion of the production of monoclonal and polyclonal antibody, the specific antibodies will be run under ELISA so that detection of the desired structural component against which the vaccine will be created can be achieved (Qiu et al., pp. 10-13).
It is an important technique which helps to identify and differentiate a specific protein from the pool of different type of foreign proteins. Many researchers has conducted immunoprecipitation followed by poly acrylamide gel electrophoresis or PAGE to determine the identity of the virus (Slonczewski and John, pp. 123-145). For the identification of the coronavirus responsible for the respiratory tract infection in humans, the protein A of Staphylococcus aureus will be used along with the specific antibody against the virus, raised in a lab rodent by injecting it several times with the virulent protein of the coronavirus. Further, the protein A along with viral lysate and specific antibody in the presence of agarose beads and the solution is incubated for the antibody-antigen interaction (Zhao et al., pp. 654-660). After the incubation period, the solution is centrifuged to separate the agarose bead precipitate from the solution and the solution is washed several times using the phosphate buffer saline solution. After this step, different gel electrophoresis and blotting techniques such as western blotting technique can be used to identify the specific protein which binds the specific antibody attached to a head of protein A. The specificity of this technique determines the effectiveness of effectiveness as without specific bindings, the gel electrophoresis will show negative results (Hsu and Katherine, pp. 1-12).
Solid phase antibody assay
The solid phase antibody assay is technique which is used to determine the virus infected serum collected from an infected human. Different researchers has utilized this technique for the detection of human affecting viruses such as Hepatitis A, Hepatitis B and many more (Reed et al., pp. 1859-1865). Therefore, in this case, for the detection of Coronavirus in affected people solid phase antibody assay can be performed. For the purpose, the micro titer plates that are coated with anti-human IgM were spread so that the antibodies generated in the human body against the virus can be captured. Thereafter, in the serum, antigens specific to coronavirus, collected from the human serum is ingested with goose erythrocytes. This leads to adsorption of erythrocyte to the antigen-antibody complexes leading to the formation of complex that attaches to the solid phase, determining the presence of Antibody IgM in the serum (Poma et al., pp. 2821-2825). Further, the separation of IgM and IgG was done with the help of rate-zonal centrifugation that helps to determine the specificity of the process. Therefore, with the detection of the IgM in the serum the solid phase antibody assay detects the presence pf virus within the serum, without using any hazardous substance such as radioactivity for the detection of Coronavirus specific antibodies in the serum (Poma et al., pp. 2825-2827).
Flow cytometry and cell sorting
This is an important technique used by researchers for clinical diagnosis and biological researches. In this method, different heterogeneous mixture of cells, infected with virus or bacteria are placed in the suspension and different laser interrogation points are passed through it (Morono et al., pp. 2848-2849). Furthermore, the light emissions are collected and correlated to understand the cell morphology and intracellular protein and gene expression. Further, it also helps to understand the cell physiology and surface. Furthermore, as the technique is about cell sorting, the researchers can also isolate cells, by diverting it from the flow and collect it in the homogeneous and viable portions (Mayle et al., pp. 27-30). Flow cytometer is one of the easy and cheap machine that helps to diagnose the protein structure attached with the infection and due to usage of advanced computers and optics in the process the reliability of the process enhances. Hence, in this case of coronavirus infection, usage of flow cytometry can be utilized to determine the structure of the virulent protein so that vaccine preparation against it becomes easier (Morono et al., pp. 2841-2848).
The Luminux assay in combination with one-step multiplex reverse transcription PCR or RT-PCR is used for the creation of Liquichip assay that helps to detect the respiratory tract infecting viruses such as influenza, hepatitis and others. However, for the detection of coronavirus, this process is not being used, therefore it is a challenge for the virus detection and vaccine preparation against the specific virus (Zhai et al., pp. 1502-1504). For this purpose, the virus serum which is collected from the infected rodent is diluted up to 10 fold and for all the dilutions were assessed through the RT-PCR technique. After the generation of the RT-PCR bands, standard curves were generated so that the samples with detection limits can be obtained for the further assessment. As the process utilizes RT-PCR technique for the identification of desired protein, the reliability of the process for the virus identification from the pool of infection is enhanced (Nejman-Fale?czyk et al., pp. 4745-4750). The time and cost efficiency is one of the major positive factors for the assessment as to complete the flow cytometric analysis and target amplification, less than 2 hours is required where as for the RT-PCR process less than 3 hours is needed. Therefore, for the detection of coronavirus in the people affected with coronavirus, the process can be utilized (Zhai et al., pp. 1499-1502).
Analysis of cell cycle and cell death
Almost all the virus, affecting humans utilizes techniques such as cell cycle arrest and apoptosis for the regulation of their infection cycle. However, there are instances where the coronavirus utilizes cell cycle perturbation and apoptosis for the enhancement in their own cell replication within the host cell. In many recent studies it was observed that the positive sense RNA of the coronavirus has the capability to arrest the cell cycle of host body such as according to Dyall et al. (pp. 4885-4887), the coronavirus that causes respiratory tract infection in humans are responsible for the arrest of G0 to G1 transition of cell cycle and the nucleocapsid is responsible for the arrest of S phase or synthesis phase of cell cycle. Therefore, this process can also be used in the detection of coronavirus that infected humans in the western region of USA (Dyall et al., pp. 4887-4893).
The methodology of this research will contain several molecular biology and immunological techniques. First the sample will be collected from infected person and one rodent in which the symptoms of coronavirus infection is observed. After that, two groups of rodents will be infected with the antigens and monoclonal and polyclonal antibodies will be prepared. These monoclonal and polyclonal antibodies will be prepared for two type of coronavirus samples, one collected from human and the other collected from rodent. Further for the detection and characterization, ELISA will be conducted. After the successful detection, consent from the government will be taken for the vaccine development and for the purpose, isolation of viral envelop and raising antibodies against the envelop will be done. Finally, the vaccine will be applied on animal and human samples so that the effectiveness of the vaccine can be calculated.
The Coronavirus emergence was first observed in the middle east country Saudi Arabia and the strain is known as MERS-CoV whereas, the other strain is known as SARS-CoV that are found to be effecting populations in western region of USA. Therefore, the assignment was determined to characterize the coronavirus infection the population and for that purpose, it utilizes the generation of monoclonal and polyclonal antibodies against the specific viral antigens. Further, using this process, the specific viral structural component that is the outer envelope was determined for the vaccine preparation. This research proposal also conducted literature review to critically determine the usage of viral envelop for the vaccine preparation. Finally the methodological flow of the research has also been presented.
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