GENOMIC STRUCTURAL IDENTIFICATION AND IMMUNOINFORMATICS STUDIES FOR SARS‐COV‐2

GENOMIC STRUCTURAL IDENTIFICATION AND IMMUNOINFORMATICS STUDIES FOR SARS‐COV‐2

Dowluru SVGK Kaladhar

Department of Microbiology and Bioinformatics, UTD, Atal Bihari Vajpayee University, Bilaspur - 495001, Chhattisgarh, India.

ABSTRACT: Coronaviruses are a group of viruses that belongs to the Coronaviridae family. These viruses have helical symmetry, enveloped, positive-sense, single-stranded RNA genome with nucleocapsid that causes diseases in birds and mammals. SARS-CoV 2 complete genome has genome size of 29,903 bp, having nine genes identified as Orf1a Polyprotein, Orf1ab Polyprotein, ORF3a Protein, ORF7a Protein, ORF8 Protein, Envelope proteins, Spike Surface glycoprotein, Membrane glycoprotein, and Nucleocapsid Phosphoprotein. Based on the ProtParam result, ORF1a has more molecular weight (489989 daltons), and Envelope protein has a less molecular weight (8365 daltons). The pI is lower in Orf1a polyprotein, Orf1ab Polyprotein, ORF3a Protein, ORF8 Protein, and Spike Surface glycoprotein) and high in ORF7a Protein, Envelope proteins, Membrane glycoprotein, and Nucleocapsid Phosphoprotein. The Orf1a Poly-protein, Orf1ab Polyprotein, ORF3a Protein, Envelope proteins, Spike Surface, and Membrane glycoprotein are stable. The higher aliphatic index and more hydrophobicity of membrane glycoprotein suggest an increase in the thermostability of protein might favor an increase in its solubility. The vaccines like EIAV vaccine gp45, anti-SARS m396 Antibody, EIAV vaccine gp45, and E protein of the Japanese encephalitis live attenuated vaccine virus are less effective than Curcumin for SARS-CoV 2. Hence better vaccines are to be developed for respiratory viruses like SARS-CoV 2 in the future.

Coronavirus, SARS-CoV-2, Genome, Immunoinformatics studies

INTRODUCTION: Coronavirus disease 2019 (COVID-19) or SARS-CoV 2 (Severe Acute Respiratory Syndrome Related Coronavirus 2) is a new emerged human coronavirus in Wuhan city of China and rapidly spread throughout the world ^{1, 2, 3}. In December 2019, the authorities of China were declared that the virus is new and emerging that may spread to all over the world. Within short period, the coronavirus was spread to more than 200 countries all around the world.

On 11-3-2020, WHO (World Health Organization) declared COVID-19 as pandemic disease, an epidemic associated with geographic spread to several countries and cause disease ⁴. Scientists and medical researchers have identified several microbial species that cause diseases like Cholera, bubonic plague, smallpox, and influenza that are the most brutal killers in human history ⁵.

In 1918 influenza virus originated in France, China, and Britain, is a severe pandemic that was occurred in the past, where more than 50 million people had died ⁶. In 1956, Asian flu was originated in China and estimated about two million deaths worldwide. In 2002 SARS (Severe acute respiratory syndrome) emerged in China ⁷, spread to 37 countries, causing global panic with more than 8,000 people infecting and about 750 people have died.

The SARS transmission after 2004 was not reported. In 2009, Swine flu or influenza (H1N1) was first identified in Mexico, with an estimated 151000-575000 people died worldwide. MERS-CoV (Middle East Respiratory Syndrome Coronavirus) first identified in Saudi Arabia spread to 27 countries infected 2400 cases and 912 deaths (4 out of 10 patients died). Like other coronavirus family members, COVID-19 is a novel mutated virus having infected cases of 5, 03, 274 with 22 342 deaths as on 26-3-2020. As on 3-8-2020, the COVID-19 virus having infected cases of 18.305.496 with 694,058 deaths worldwide. The world is commonly called this virus Coronavirus. About 70% of people infected are recovered and developed herd immunity. Hence the emerging viruses originated previously from China, US and Saudi Arabia may be due to eating habits, mobile radiations, advanced microbial researches or climate factors. Coronaviruses (CoVs) cause gastrointestinal and/or respiratory disease/s in humans, cattle, swine, and poultry ⁸. The effect of Coronaviruses in chickens and humans are upper respiratory tract infections. In cows and swine, the Coronaviruses cause diarrhea.

The Coronavirus is a viral genetic material with lipoprotein present as a particle outside host machinery ⁹. It contains a replicative protein nsp ⁹ acts as a single-stranded RNA-binding subunit that is distinctive in the world of RNA virus ¹⁰. When the virus enters into living cells, it multiplies fast using host machinery within human cells. The virus reaches into the human system through the nose or mouth or eyes or ears and finally reaches the alimentary canal, blood, liver and lungs. In lungs, the epithelial cells containing ACE2 receptor joins with spike glycoprotein of Coronavirus and kills all epithelial cells in lungs ^{11, 12}. Within seven to ten days, millions of cells present in human system will be damaged and then coronavirus acts on the Immune system. The genetic material may modify into other forms, infect other parts of the host complex system and may damage complete system.

Humans with a good immune system can be isolated, quarantined/ recovered ¹³. The Immune system will protect humans from foreign bodies by passive immunity. If the immune system disturbs, it causes severe effects like dry cough, cold, inflammation, and fever.

Some of the T-cells, with the help of proteins called cytokines, come closer to coronavirus and become non-effective ¹⁴. The resources of host machinery will be used very fast, and coronavirus multiplies rapidly and kills Neutrophils and Killer T-cells. In some cases, the Neutrophils and Killer T cells act as toxic and make other human cells to be self-Killed. If cells in the lungs are killed, it leads to fibrosis and respiratory problems. In some humans with good immune system regains its strength of Neutrophils and Killer T cells and dominates coronavirus, and stop infecting other cells.

In some cases, there will be critical cases that are hidden at present conditions. There may be a flu-like infection that causes pneumonia (due to damage of alveoli), where the patient should be kept in ventilators and should supply oxygen ¹⁵. If the virus spreads to the entire body and controls other bacteria in humans and cells, the patient will die. Quantitative comparisons of quarantine, case identification and isolation, infection control precautions, and immunization interventions are the effectively controlling measures for viral diseases ¹⁶.

The slow pandemic viruses like HIV are not that much dangerous as it spreads slowly to the people. The fast Pandemic species like Coronavirus is more dangerous that kills many people and stays for longer periods ¹⁷. The facilities like vaccines, drugs, ventilators, and manpower become less, and many people will have died. To control this situation, the fast-spreading viruses should be converted into slower pandemic species. The infected people should be isolated and treated in the first condition, and other healthy humans should stay away. As there is no proper vaccine in the present condition, human behavior, distance maintenance, and prevention measures can only make fast pandemic coronavirus slow pandemic coronavirus ¹⁸.  The hands should be washed with soap (destroy lipid envelop) and sanitizers. The virus may persist on glass, paper, plastic, and wood for 4 days, Aluminum and surgical gloves for 8 days, and steel for 48 days. Like other coronaviruses like SARS, COVID-19 has been predicted to be transferred to humans from animals like Bats ¹⁹. The most common symptoms of COVID-19 are tiredness, fever, and a dry cough, sometimes with a runny nose, sore throat, nasal congestion and aches and pains or diarrhea. The elderly people having medical problems like diabetes, high blood pressure, heart problems, or chronic respiratory conditions are having greater risk of serious illness and deaths from COVID-19. The virus is stable for several hours to days in aerosols and on surfaces moving smaller distances as particles. The virus can live two to three days on plastic and metals. Immunoinformatics helps in the development and design of novel or enhanced vaccines that target through in-silico genome analysis that has great potential to fight against diseases. Several human types of HPV were identified, but vaccines for few types have only developed. The newly emerged virus COVID-19 has several types hence success rate for the development of vaccines may be less.

Antibiotics are of no use, antiviral drugs will not work, and there is currently no vaccine for Coronavirus. Recovery from this disease depends on the strength of the immune system of humans. The present work provides a better understanding of the genome and immune control activity by vaccination using in silico methods.

MATERIALS AND METHODS:

Genome Retrieval: The SARS CoV 2 complete genome sequence is retrieved from the NCBI database with Accession number NC_045512.2 isolate from Wuhan-Hu-1. The genome was analyzed for genomic and immunoinformatics studies for better relevant vaccination.

Gene Prediction: The gene prediction for SARS CoV 2 complete genome was predicted using FGENESV0, a prediction of potential genes in viral genomes server. The server predicts the genes present in the viral genome.

Gene Identification: The gene identification for the protein sequences of SARS CoV 2 was obtained using FGENESV0 is submitted to BLASTp. Based on the predicted alignment scores and the function of proteins can be identified for a better understanding of the character of the virus.

Characterization of Proteins: The proteins present in SARS CoV 2 are characterized using the ProtParam tool present in ExPASy server. The protein characters like Molecular weight, Theoretical pI, estimated half-life, instability index (II), Aliphatic index, and Grand average of hydropathicity (GRAVY) for Coronavirus have been predicted in this method.

Prediction of Antigenic Epitope Regions: Epitope regions in the viral genes have been conducted using the SVMTriP server. The prediction may help the design of vaccine components in recognition of antibodies in cells.

Properties of Computer: The system properties conducted in the present work is as follows:

1. Window Edition: Windows 10 Home
2. Processor: Intel Celeron CPU N3350 of 1.10GHz with RAM of 4GB and x64-based processor

Docking Studies: The modeled structure for Spike glycoprotein of SARS CoV 2 was designed using SWISS-MODEL. The Spike protein (Antigen) was taken as a receptor, and 2O8Y (PDB file) of ACE2 (Antibody) as ligand are selected to check for Antigen-Antibody activity.

The vaccine structures 2g75 (anti-SARS m396 Antibody), 3wmj (EIAV vaccine gp45), 5mv2 (E protein of the Japanese encephalitis live attenuated vaccine virus) were retrieved from Protein DataBank (PDB) and Curcumin E (Designed and optimized by ChemSW software) are used as ligands for Spike glycoprotein. Docking studies are conducted using Hex v8.0.0.

RESULTS:

Genome Retrieval: The SARS CoV 2 genome sequence of NC_045512.2 related to severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome has been retrieved from NCBI. The genome has 29903 bp, ss-RNA with linear structure. As per the genomic data, SARS CoV 2 is a novel emerging coronavirus associated with a severe respiratory disease emerged in Wuhan of Hubei province, China. The virus is formerly called 'Wuhan seafood market pneumonia virus’.

Gene Prediction: The gene prediction for the retrieved SARS CoV 2 genome was predicted using FGENESV0, a Prediction of potential genes in viral genomes server. The number of predicted genes in the 29903bp length genome is nine, and the details are shown in Table 1.

TABLE 1: GENE PREDICTION FOR SARS COV 2

Gene Identification: The identification of SARS CoV 2 genes using BLASTp was shown in Table 2.

TABLE 2: GENE IDENTIFICATION OF SARS COV 2

The structure of the identified SARS CoV 2 was shown in Fig. 1

FIG. 1: SARS COV 2 PHENOTYPIC AND GENOMIC STRUCTURE

TABLE 3: PROTEIN CHARACTERIZATION OF SARS COV 2

Characterization of Proteins: The proteins are characterized using the ProtParam tool was shown in Table 3. Table 3 shows that ORF1a has more molecular weight (489988.91), and the Envelope protein has a less molecular weight (8365.04). The pI (Isoelectric point) is the pH at which protein does not migrate in electric field. The antigens with lower pI generally have tissue uptake and a longer half-life. For acidic proteins, the pI will be lower (Orf1a Polyprotein, Orf1ab Polyprotein, ORF3a Protein, ORF8 Protein, and Spike Surface glycol-protein), and for basic proteins, pI will be high (ORF7a Protein, Envelope proteins, Membrane glycoprotein, and Nucleocapsid Phosphoprotein). Orf1a Polyprotein, Orf1ab Polyprotein, ORF3a Protein, Envelope proteins, Spike Surface, and Membrane glycoprotein are stable. The higher aliphatic index (Membrane Glycoprotein) suggests an increase in the thermostability of protein might favor an increase in its solubility. In the GRAVY result, a more positive score present in Membrane Glycoprotein indicates more hydrophobicity.

TABLE 4: ANTIGENIC EPITOPES FOR ORF1A POLYPROTEINS OF SARS COV 2

* The epitopes recommended are labeled by the flags

TABLE 5: ANTIGENIC EPITOPES FOR ORF1AB POLYPROTEIN OF SARS COV 2

* The epitopes recommended are labeled by the flags

TABLE 6: ANTIGENIC EPITOPES FOR SPIKE SURFACE GLYCOPROTEIN OF SARS COV 2

* The epitopes recommended are labeled by the flags

Prediction of protein surface regions that are preferentially recognized by antibodies (antigenic epitopes) can help the design of vaccine components and immunodiagnostic reagents ₂₀.

A method to predict Linear antigenic epitopes is conducted by Support Vector Machine (SVM) has been utilized by combining the Tri-peptide similarity and Propensity scores (SVMTriP).

The antigenic epitopes for Orf1a polyprotein, Orf1ab polyprotein, Surface glycoprotein, ORF3a protein, Membrane glycoprotein, ORF7a protein, ORF8 protein, and Nucleocapsid Phosphoprotein were shown from Table 4 to 11. There are no antigenic epitopes found in the Envelope protein of SARS CoV 2.

TABLE 7: ANTIGENIC EPITOPES FOR ORF3A PROTEIN OF SARS COV 2

* The epitopes recommended are labeled by the flags

TABLE 8: ANTIGENIC EPITOPES FOR MEMBRANE GLYCOPROTEIN OF SARS COV 2

* The epitopes recommended are labeled by the flags

TABLE 9: ANTIGENIC EPITOPES FOR ORF7A PROTEIN

* The epitopes recommended are labeled by the flags

TABLE 10: ANTIGENIC EPITOPES FOR ORF8 PROTEIN OF SARS COV 2

* The epitopes recommended are labeled by the flags

TABLE 11: ANTIGENIC EPITOPES FOR NUCLEOCAPSID PHOSPHOPROTEIN OF SARS COV 2

* The epitopes recommended are labeled by the flags

There are more antigenic sites in Orf1a polyprotein, Orf1ab polyprotein, Surface glycoprotein, and Nucleocapsid phosphoprotein of SARS CoV 2. As per the previous studies, it was found that the Coronavirus attaches to a receptor on respiratory cells called ACE2 (angiotensin-converting enzyme 2). The small molecular key present on SARS-CoV-2 that gives an entry of the virus into the host cell is called a spike protein, or Surface Protein, or S-protein.

TABLE 12: DOCKING RESULT OF ACE2 WITH SPIKE PROTEIN OF SARS COV 2

As per the previous information, lesser the binding energy may be considered as a good inhibitor of Spike Glycoprotein of coronavirus ^{21, 22}. Hence, the activity of Curcumin E is more on Spike protein of SARS CoV 2. The vaccines like EIAV vaccine gp45, anti-SARS m396 Antibody, EIAV vaccine gp45, and E protein of the Japanese encephalitis live attenuated vaccine virus are less effective than Curcumin for SARS CoV 2  Table 12 and Fig. 2. Hence, better vaccines are to be developed for SARS CoV 2.

FIG. 2: DOCKING RESULT OF SPIKE GLYCOPROTEIN OF CORONAVIRUS WITH SELECTED MOLECULES

DISCUSSION: As on 23-3-2020, there are 358803 Coronavirus Cases, 15,433 Deaths, 100,645 Recovered and 242,725 active cases. The linear graph of the growth has been shown in Fig. 3.

FIG. 3: TOTAL CASES AND DEATHS OF SARS-COV-2 TIMELINE AS ON 23-3-2020

As of 25-3-2020, there are 434654 coronavirus cases, 19604 deaths, 111854 recovered, and 303196 active cases. As on 12-10-2020, there are 37,754,464 Coronavirus Cases, 1,081,500 Deaths, 28,361,239 Recovered and 8,311,725 active cases.  The linear graph of the growth has been shown in Fig. 4.

FIG. 4: TOTAL CASES AND DEATHS OF SARS-COV-2 TIMELINE AS ON 12-10-2020

The temperatures on 25-3-2020 in some infected places are shown in Table 12. There may be no effect of viral survival and activities based on temperatures but may be active in rainy and winter seasons Table 13. The data showed that the origin country of SARS-CoV-2, China, has controlled infections and deaths, and the methodology of control may not be followed by other countries. There may be few more microbial origins in the future that may damage economic and government strategies, which may be dangerous for the human community.

TABLE 13: INFECTIONS, DEATHS AND TEMPERATURES IN SOME COUNTRIES BY SARS-COV-2

Most humans follow ancient and modern methods of health care products and medicines to keep the health fit with strong immunity ^{23, 24, 25}. Most of the techniques followed by humans in the past will make a better health habit for future generations. The activity of Curcumin E is more on Spike protein of SARS-CoV-2 compared to selected vaccines. The vaccines like EIAV vaccine gp45, anti-SARS m396 Antibody, EIAV vaccine gp45, and E protein of the Japanese encephalitis live attenuated vaccine virus are less effective than Curcumin for SARS-CoV-2. Currently, there is no effective drug treatment or vaccine exists for the treatment of SARS-CoV-2. Aging diseases like diabetes, cardiovascular disease, hypertension, chronic respiratory disease, and cancer are showing a high risk of death due to SARS-CoV-2 ²⁶.

Remdesivir that is developed for the treatment of viruses like the Ebola virus and Marburg virus is effective against SARS-CoV-2. Hence, better vaccines are to be developed in the future.

CONCLUSION: The viruses that emerge from time to time can spread to several countries and can make pandemics.

Hence the immunity has to be developed, and vaccine development procedures have to be established for the control of novel viruses.

ACKNOWLEDGEMENT: The author would like to thank the administration of Atal Bihari Vajpayee University and all the staff for their assistance.

CONFLICTS OF INTEREST: There is no known conflict of interest associated with the publication

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    How to cite this article:

    Kaladhar DSVGK: Genomic structural identification and immunoinformatics studies for SARS‐COV‐2. Int J Pharm Sci & Res 2021; 12(4): 2117-25. doi: 10.13040/IJPSR.0975-8232.12(4).2117-25.

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