Vishal Trivedi^1*, Kush Biswas², Santosh Fattepur³ and Nagaraja Sreeharsha^4,5

¹Department of Pharmacy, Madhav University, Abu Road, Pindwara, Rajasthan, India

²Department of Pharmaceutical technology (Biotechnology), National institute of Pharmaceutical Education and Research, Mohali, sector 67, SAS Nagar, Punjab, India

³School of Pharmacy, Management and Science University,Seksyen 13, 40100 Shah Alam, Selangor, Malaysia

⁴Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa-31982, Saudi Arabia.

⁵Department of Pharmaceutics, Vidya Siri College of Pharmacy, Off Sarjapura Road, Bengaluru - 560 035, Karnataka, India

Corresponding Author Email: vishaltrivediqa@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/2080

Abstract

In Present time Novel coronavirus (SAR-CoV-2) was the biggest outbreak on human kind. SARS recognized febrile respiratory illness. It was first discovered in southern China in November 2002, and spread all other countries.Now SARS-CoV-2 was also originated from Wuhan, china. It was transmitted from human to human. For developing a molecular diagnostic for 2019-nCoV a PCR detection assay was developed. Sanger, Illumina, and Oxford nanopore techniques are used for sequencing analysis for 2019-nCoV.Based on different types of data base it was concluded that the SARS-CoV-2 was less mutated than other seasonal flu. This study shows the complete genome sequence and characteristic of SAR-CoV-2. SAR-CoV-2 has 29,903 nucleotides in length which is quite similar to others coronavirus. A complete genome sequence of different-different countries was studied.

Keywords

Coronavirus (SAR-Cov-2); Complete Genome Sequencing And Characterizations; PCR Detection

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Copy the following to cite this article: Trivedi V, Biswas K, Fattepur S, Sreeharsha N. Study on Genome Sequence of Novel Corona virus (Sars-Cov-2) Strains in Different Countries. Biomed Pharmacol J 2020;13(4).

Copy the following to cite this URL: Trivedi V, Biswas K, Fattepur S, Sreeharsha N. Study on Genome Sequence of Novel Corona virus (Sars-Cov-2) Strains in Different Countries. Biomed Pharmacol J 2020;13(4). Available from: https://bit.ly/3aFO9TC

Introduction

2019-nCoV are large about 120 to 160nm long, spherical particles with a linear, non-segmented, capped, and polyadenylated positive-sense.^1,2 It is a single-stranded RNA genome that isencapsulated in a helical nucleocapsid. The envelope is made up of intracellular membranes and have a characteristic crown of widely spaced club-shaped spikes that are 12 to 24 nmlong.^3,4 Based ongenetic and serological relationships corona virus can be classified into three groups.^5,6

Table 1: Classification of Corona virus.

Group 1	Group 2	Group 3
Porcine epidemic diarrhea virus (PEDV) Porcine transmissible gastroenteritis virus (TGEV) Canine coronavirus (CCoV) Feline infectious peritonitis virus (FIPV) Human coronavirus 229E (HCoV-229E) Human coronavirus NL63 (HCoV-NL63)	Murine hepatitis virus (MHV) Bovine coronavirus (BCoV) Human coronavirus OC43 (HCoV-OC43) Rat sialodacryoadenitis virus (SDAV) Porcine hemagglutinating encephalomyelitis virus (PHEV) Canine respiratory coronavirus (CRCoV) Equine coronavirus (ECoV)	Avian infectious bronchitis virus (IBV) Turkey coronavirus(TCoV)

The corona virus genomes are the largest of the known RNA viruses (27 to 31.5 kb) and are polycistronic, generating a nested set of sub- genomic RNAs with common 5 and 3 sequences.^7,8 The 5 two-thirds of the genome consists of two large replicase open reading frames (ORFs), ORF1a and ORF1b. The ORF1a polyprotein (pp1a) can be extended with ORF1b-encoded sequences via a 1 ribosomal frame shift at a conserved slippery site, generating the 7,000-amino-acid polyprotein pp1ab, which includes the putative RNA-dependent RNA polymerase (RdRp) and RNA helicase (HEL) activity.^9,10,11

Virus isolation

A Pathogen free human airway epithelial (HAE)cells were used for virus isolation.¹² Briefly, bronchoalveolar lavage fluids or throat swabs from the patients were inoculated into the HAE cells through the apical surfaces.HAE cells were maintained in an air–liquid interface incubated at 37°C. The cells were monitored daily for cytopathic effects by light microscopy and the cell super natants were collected for use in quantitative RT-PCR assays.^13,14,15 After three passages, apical samples were collected for sequencing.¹⁶

Genome Sequences of SAR-CoV-2

The genome of SARS-CoV-2 is a 29,903 nucleotide, polyadenylated RNA, and 41% ofthe residues are G or C.¹⁷ The genomic organization of corona viruses, having the characteristic gene order [5 replicase (rep), spike (S), envelope (E),membrane (M), and nucleocapsid (N)-3] and short untranslated regions at both termini.^18,19 The SARS-CoV-2 rep gene,which comprises approximately two-thirds ofthe genome, is predicted to encode two polyproteins ORF1a and ORF1b that undergo cotranslational proteolytic processing.²⁰ Downstream of four open reading frames (ORFs)are used to encodethe structural proteins S, E, M, and N, which are common to all known corona viruses.^21,22 Hemagglutinin – esterase, are present between ORF1b and S of corona viruses. These corona viruses encoded non-structural proteins which are located between S and E, between M and N, or downstream of N.^23,24The genome of SARS-CoV-2 contains ORFs for five potential non-structural proteins that are more than 50 amino acids long inthese intergenic regions.²⁵ overlapping of ORFs encoding show the proteins of 274 and 154 amino acids are located between S and E. Three non-structural genes, X3, X4, and X5 are located between M and N.^26,27 GenBank database (BLAST and FastA) show that there is no significant sequence similarity between these potential non-structural proteins ofSARS-CoV-2 and any other proteins.^28,29 ORFs encoding proteins are more than 50 amino acids long in the structural genes of SARS-CoV-2 (such as N, S, and rep).^30,31SARS-CoV-2 repgene products have genomic RNA, and the remaining viral proteins are translated from sub genomicm RNAs that form a 3-coterminal nested set,each with a 5-end derived from the genomic 5 leader sequence.^32,33Sub  genomicm RNAs are synthesized from a discontinuous transcription process. The SARS-CoV-2 leader sequence was mapped by comparing the sequence of rapid amplification of cDNA ends.^34,35The products which are synthesized from the N gene mRNA with those synthesized from genomic RNA.³⁶ A sequence, AAACGAAC (genomic nucleotides 65to 72), was identified where the N gene mRNA and genomic sequences diverged.³⁷ This sequence was also present upstream of ORF1a and immediately up stream of five other ORFs suggesting that it functions as the conserved core of the transcription-regulating sequences (TRSs).^38,39 The nucleotides required for TRS function must be identified experimentally.⁴⁰ The production of sub genomic mRNAs of SAR-CoV-2 proposes that discontinuous transcription has occurs during the synthesis of negative strand. Sub-genomic negative strands containing a complimentary copy of the leader sequence at their 3 termini serve as templates for synthesis of sub-genomic mRNAs.^41,42 At the site of 5 terminus of the genome, the TRS conserved core sequence appears six times in the remainder of the genome.⁴³ The positions of TRS in the genome of SARS-CoV-2 predict that sub-genomic mRNAs of 8.3, 4.5, 3.4, 2.5, 2.0, and1.7 kb, not including the poly(A) tail. At least five sub-genomic mRNAs were detected by hybridization of RNA from SARS-CoV–2 infected cells.^44,45 Sizes of the five predominant bands correspond to the sizes of five of the predictedsub genomic mRNAs of SARS-CoV-2. Full-length of genomic RNA was not detected, probably because it is the least prevalent viral RNA in infected cells.^46,47 The predicted 2.0-kb transcript was also not detected, which suggests that the consensus TRS at 27,771 to 27,778 is not used or that it is a low-abundance mRNA.⁴⁸ Byanalogy with other corona viruses, the 8.3-kband 1.7-kb sub-genomic mRNAs are predicted to be monocistronic, directing translation of S andN, respectively, whereas multiple proteins could be translated from the 4.5-kb,3.4-kb and 2.5-kb mRNAs. It is possible that the 3.4-kb band contained more than one mRNA species that was not resolved in the gel or that the monocistronicm RNA.^49,50,51

CDC sequencing strategy

The whole-genome sequences of SAR-CoV-2 were generated by a combination. Sanger, Illumina, and Oxford nanopore sequencing were used for this detection.⁵² In this CDC sequencing strategy at First viral RNAs were extracted directly from clinical samples with the QIAamp Viral RNA Mini Kit, and then used to synthesise cDNA with the SuperScript III Reverse Transcriptase and N6 random primers, followed by second-strand synthesis with DNA Polymerase I, Large Fragment.^53,54 Viral cDNA libraries were prepared with use of the Nextera XT Library Prep Kit (Illumina), then purified with Agencourt AMPure XP beads followed by quantification with an Invitrogen Qubit 2. 0F luorometer.^55,56 The resulting DNA libraries were sequenced on either the MiSeq or iSeq platforms (Illumina) using a300-cycle reagent kit. About 1·2–5 GB of data were obtained.⁵⁷ The raw fast Q files were filtered then subjected tode novo assembly with the CLCBio software. Mapped assemblies were also done using the bat derived SARS-like corona virus isolate bat-SL-CoVZC45 as a reference. Variant calling, genome alignments, and sequence illustrations were generated with CLCBio software, and the assembled genome sequences were confirmed by Sanger sequencing.^58,59Rapid amplification of cDNA ends (RACE) was doneto obtain the sequences of the 5ʹ and 3ʹ termini, using the Invitrogen 5ʹ RACE System and 3ʹ RACE System, according to the manufacturer’s instructions.⁶⁰ Gene-specific primers for 5ʹ and 3ʹ RACE PCR amplification were designed to obtain a fragment of approximately 400–500 bp for the two regions. Purified PCR products were cloned into the pMD18-T Simple Vector and chemically competent Escherichia coli. PCR products were sequenced with use of M13 forward and reverse primers.^61,62,63

Development of molecular diagnostics for 2019-nCoV

COVID-19 disease is spreads all over the world. IAEA and FAO (Food and Agriculture Organization)are supporting the most important laboratory technique Real time reverse transcription–Polymerase Chain Reaction (real time RT-PCR)for detecting, tracking, and studying of COVID-19. ^64,65,66

On the basis of the genome sequences obtained, a real-time PCR detection assay was developed. PCR primers and probes were designed using Applied Bio systems Primer Express Software on the basis of our sequenced virus genomes. Primers and probes were synthesised by BGI. RT-PCR was done with an Applied Bio systems 7300 Real-Time PCR.^67,68 It is a nuclear-derived method for detection of genetic material from any pathogen. Radioactive isotope markers are used to detect targeted genetic materials. Fluorescent dyes were used to replace the isotopic labelling. While real time RT-PCR is now the most widely used method for detecting corona viruses in many countries.^69,70,71(SARS-CoV-2) only contains RNA. In SARS-CoV-2 RNA take control and reprogrammed the cells so that they multiple and make more viruses. SAR-CoV-2 detected in the body using real time RT-PCR. In which a sample is taken from infected person body like nose or throat.^72,73,74 The taken sample is treated with several chemical solutions that remove substances,only the RNA is been extracted from the taken sample.The RNA is reverse transcription process. By using specific enzymes this RNA was converted into DNA. short fragments of DNA are added that are complementary to specific parts of the transcribed viral DNA. These fragments of DNA to target sections of the viral DNA.^75,76,77 The genetic fragments are for building DNA strands during amplification, while the others are for building the DNA and adding marker labels to the strands, which are then used to detect the virus. These mixers then placed in a RT-PCR machine.^78,79 The machine cycles heat and cool the mixture chemical reactions this create new, identical copies of the target sections of viral DNA. This cycle can repeat over and over to continue copying the target sections of viral DNA. Each cycle doubles the amount: two copies become four, four copies become eight, and so on.^80,81 A standard real time RT-PCR setup goes through 35 cycles, which means that by the end of the process, around 35 billion new copies of the sections of viral DNA are created. the marker labels and a fluorescent dye were used to measured.^82,83 Computer’s has been used for tracking the amount of fluorescence in the taken sample after each cycle. If the amount of fluorescence goes over a certain level, it gives the conformation of virus present.⁸⁴ This technique is highly sensitive and specific. It can deliver a reliable diagnosis as fast as three hours. by Comparing with other available virus isolation methods, real time RT-PCR is much faster and it has a lower potential for contamination or errors. the entire process can be done within a closed tube. It is the most accurate method available for detection of the SAR-CoV-2.^85,86,87

Effects of Genome sequence COVID-19 on different countries

Table 2: Effect of different genome sequences on different countries

Countries	Total cases	Active cases	Total recovered	Total death
Europe	1,322,784	7,36,539	4,59,814	1,26,431
North America	1,120,597	8,79,941	1,76,244	64,412
Asia	4,95,108	2,30,909	2,46,272	17,927
South America	1,55,592	93,708	54,294	7,590
Africa	35,779	22,383	11,868	1,528
Oceania	8,314	1,274	6,933	107

This study shows the effects on changing in   genomic sequences of COVID 19 on different countries. It was stated from Wuhan city, china and spread all over the world. In December 2019, an outbreak of apparently viral pneumonia of unknown ethology emerged in the city of Wuhan, in the Chinese province of Hubei. Chinese scientist done 7,700 genome sequences of COVID-19. These genome sequences help to vital tracking, how the virus mutates time by time as it spread and also used for development of diagnostic tests and vaccines.⁸⁸

Figure 1: Genomic sequence done on COVID19 in different countries Click here to View figure

Data shows that Africa and south America has only 1% of genome sequencing done on COVID-19. Whereas Europe and north America has the highest genome sequencing done. There should be more genomic data from Africa and south America for vaccine development.⁸⁹

9 January, 2020, health authorities of Chinese and the World Health Organization (WHO) found and announces the novel corona virus, known as SAR-CoV-2, these agents responsible for the pneumonia cases. On 11 and 12 January, the health authorities of Chinese shared the full genome sequence of SAR-CoV-2. The genome sequences of SAR-CoV-2 is crucial for the development of specific diagnostic tests and the identification of potential treatment options. Chinese scientists claim there are two main strain of SAR-CoV-2 in humans, indicating that the virus is mutating.Researchers of china say the COVID-19 virus, which has since been renamed SARS-CoV2, has evolved into two major lineages, known as “L” and “S” types.The newer and more aggressive L type strain accounted for about 70 per cent of the analysed cases, the researchers said, while the rest were linked to the older S type version.^90,91

On Friday 24 January, 2020, detection of the corona virus was confirmed in France.

India researchers [National Institute of Virology (NIV), Pune and Gujarat Biotechnology Research Centre (GBRC)] decoding the entire genome sequence of the novel corona virus, and confirmed that there are three new mutations has occurred.These viruses mutate in order to adapt to and survive in different situations. It was mutated when it will try to control by medicines. This virus is mutating fast. After establishing the genome sequence then analyse the genome in them and after that formulate a strategy to fight the virus.

World Health Organization, confirmed that the diagnosis of COVID-19 should be done by RT-PCR or gene sequencing.⁹² The RT- PCR test is a highly sensitive. It is a gene based diagnostic test. It is much more reliable and much better than the conventional test.In united states of America, Researchers have examined the genomes sequences of corona virus say the similarities between the cases suggest that the virus may have been spreading in the state for weeks. On 20 January, by the analysis of genome sequences, centre of disease control announced that Washington had the United States’ first confirmed case of corona virus. Peoples who live in the same county, but did not known to have any contact with one another. By the study of genomic sequences, it has been concluded that the virus has been spreading through other people in the community.U.S.A. scientist also concluded that these viruses are independently would arrive in the same geographical area and be genetically related unless they were connected.⁹³

Figure 2: Coronavirus death in top ten countries. Click here to View figure

In united states death rate is very much high as compared to another countries. In USA, Human-to-human transmission has been reported too much.On 27 February, the first case of community spread in the US was reported, a person who did not have any travel history to the corona virus-affected regions.New York has reported the highest number of corona virus cases and deaths in the US and also accounts for 40% of deaths in the country. The government announced a disaster declaration for the state and the National Guard has been activated.The US passed a bill on 13 March making corona virus tests free for its residents.The CDC developed the RT-PCR diagnostic panel for testing patient specimens for COVID-19. It was approved for emergency use by the U.S. Food and Drug Administration (FDA) under Emergency Use Authorization (EUA).The CDC later announced that it would use its existing seasonal influenza surveillance system for testing corona virus cases in five states including Los Angeles, San Francisco, Seattle, Chicago, and New York.^94,95

India has recorded 78,194 confirmed cases, 2,551 deaths, 26,400 cured cases, according to the latest update released by the Union Ministry for Health and Family Welfare.

Covid-19 is not easy to diagnose. Real Time-PCR can be used for virus testing followed by gene fingerprinting.There are 15 labs in India were the test has been done so far and Nineteen will be made operational as soon as possible. So far, specimens of suspected novel corona virus patients are tested at the National Centre for Disease Control (NCDC) in Delhi, Indian Council of Medical Research (ICMR)’s laboratories at Alappuzha, Bengaluru, Hyderabad and Mumbai, besides the National Institute of Virology (NIV), Pune. Under the guidance of NIV these labs are worked.In this test, a nucleic acid amplification-based assay called polymerase chain reaction (PCR) are used and a more sensitive form called reverse-transcription polymerase chain reaction(RTPCR).According to recommendations by the World Health Organization. COVID-19 diagnosis must be confirmed by RT-PCR or gene sequencing for respiratory or blood specimens. It is much more reliable and much better than the conventional test.⁹⁶

China take immediate precautions to stop the spread of the virus and provide diagnosis and treatment in-time for reduction the impacts.On 23 January, the Chinese Civil Aviation Administration guided foreign airlines to reduce their scheduled flights to Wuhan, when necessary, to prevent and control the spread of pneumonia caused by corona virus. Chinese’s government take a number of steps to stop the outbreak, which are: –

Pooling trained medical personnel from across the country

Ensuring supplies to the most vulnerable and affected areas

Construction of new temporary coronavirus hospitals on a war-footing

Extending holidays for schools and businesses as well as the Chinese Lunar New Year holiday

Researchers has allowed for gene testing, epidemic control and research institutions to use Linear Fold, its RNA structure algorithm that will help to understand the virus and screening compounds in less than half-a-minute compared to approximately an hour earlier. Bangkok (Thailand) is currently at risk from a spread of the virus. Hong Kong (China) is second on the list, followed by Taipei (Taiwan, the Republic of China).

The most ‘at-risk’ countries or regions worldwide are Sydney, New York, London, Thailand, Japan and Hong Kong. USA, Australia and the UK. In mainland of China, the cities of Beijing, Guangzhou, Shanghai, Chongqing, Guangdong, Zhejiang, Sichuan and Henan are all identified as high-risk by the researchers. A common factor behind the deaths recorded in china was smoking, those who with already damaged lungs are more likely to succumb to pneumonia. According to WHO, Italy has been hit so badly, because of the age of its population. The country has the oldest average age in Europe, second oldest in the world and corona virus disproportionately affects the elderly, whose immune system may not strong enough to stave off the pneumonia the virus causes.^97,98

First confirmed cases in Brazil came from Italy. On 28 January, the first Brazilian sequence closely resembles to the sequenced in Germany and it was different from the genome sequenced in Wuhan, the epicentre of the epidemic in China. This virus mutates relatively little, once a month on average, so there’s no point in sequencing small pieces of the genome. on February 29, the researchers isolated the sequence of whole genome of the corona virus from the second Brazilian patient diagnosed with COVID-19. Completed only 24 hours after confirmation of the case in Brazil, the study shows that there are differences between the genomes of the viruses isolated from the first and second patients. “The second genomic sequence of Brazilian is more similar to the genome sequenced in the United Kingdom, and it both differ from the Chinese sequences. This show that the COVID-19 epidemic is maturing in Europe in the sense that internal transmission is already occurring in European countries.

The main advantage of real-time epidemic monitoring. It is used to exactly identifying,where the virus came from. This information is useful to guide containment actions and help reduce dissemination of the disease. on February 26, the first case of COVID-19 was diagnosed in Brazil by molecular biology.^99,100

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