GENOME ORGANIZATION OF SARS-CoV-2 (Covid-19)

GENOME ORGANIZATION OF CORONAVIRSES

INTRODUCTION

Family coronaviridae mostly contain large enveloped viruses which are further said to be single stranded and their nature is as RNA viruses. Their genome ranges between 25-32 kb and these are the largest RNA viruses with diameter of 118-136 nm(1,2) . Large spike glycoprotein is present in them which extends up to 16-21 nm and their characteristic virion is spherical(3). Two subfamilies of this family are present, known as coronavirinae and torovirinae.(4)

Coronaviruses have largest positive-sense RNA genome. The viruses of human and other vertebrates(5). These are the most critical pathogens as they can effect birds, mouse, bats, livestock and human beings. Many wild animals are also affected by coronaviruses (CoVs). There are certain outbreaks in the past such as, in 2002-2003 severe acute respiratory syndrome and in 2012 Middle east respiratory syndrome was observed. Both of these SARS and MERS reflects that this virus can be transmitted from animal to human and even human to human(6). At that time they were regarded as the emerging CoVs. Most recent outbreak of coronavirus has been observed in Wuhan in December 2019. It appears as the most mysterious outbreak in the history of the human world, and it grabs the attention of whole world towards it. Since the recognition of this, etiological studies have been conducted and many researchers are trying to control the outbreak.(7,8)

In the start, this was thought as a mysterious pneumonia but later on five Chinese laborites confirmed that it is actually novel coronavirus (nCoV), as it was the temporary name given by the World Health Organization on 12 Jan. 2019. Such kind of sudden outbreaks reminds us that Global health is at a threat due to these CoVs. As our climate and ecology are continuously changing and moreover human and animal interactions are being increased day by day, we are at more risk to handle such more outbreaks in the future which may either from CoV or from other sources. We

are at an urgent need to develop, at least , therapeutic strategies against these CoVs and to have effective vaccines to cure these.(9)

CoV PATHOGENESIS

CoVs show tissue tropism, like mammals are affected by alpha and beta-coronavirus while birds are fish are infected by gamma and delta-coronavirus. Later can also infect mammals. Before 2019 HCoV-NL63, HCoV-229E, HKU1 and HCoV-OC43 and SARS-CoV along with MERS-CoV were the CoVs that can cause diseases in humans. Out of these 6, first four can cause upper mild respiratory diseases. They can also cause severe infections as well. The later two can cause lower respiratory diseases and also SRS in humans. CoVs can also cause economic loss by infecting wild animals and mice, bats etc. (10,11)

HKU2-related CoV, causes diseases in pigs and more than 24000 were dead during this outbreak, this was given the name of swine-acute diarrhea syndrome, CoV. This was the first case reported due to the involvement of bats. A beta coronavirus, 2019-nCoV is the newest outbreak to recorded in human society in Wuhan, China. This is even more mild than SARS & MERS as it can cause lower respiratory tract and also pneumonia in humans. This virus is transferred from animals to humans. Human to human transmission is also observed. (12)

GENOME STRUCTURE AND REPLICATION

As described earlier, CoVs belongs to subfamily coronavirinae and the family is coronaviridae. The order of these viruses is Nidovirales. Four genera are included in this alpha, beta, gamma and deltacoronavirus. Their genome contains 5’ cap structure and 3’ poly A tail, these are single stranded RNA with positive sense (+ssRNA) base pair also different and may be they are up to 30kb. The RNA is of much use especially genomic RNA, mostly it is used as a template which works to translate most polyproteins 1a/1ab(13). These polyproteins are useful in formation of replication-transcription complex as they encode for structural proteins. All this is happening in double membrane vesicle.

In the next step, a group of nested subgenomic RNA will be synthesized in a discontinuous manner by RTC, the major feature of these RNAs is that they contain the same 5’ leader and 3’ terminal sequences. At transcription regulatory sequences, transcription termination takes place, and these sequences are present between different open reading frames. There are also some minus-stranded sgRNAs which serves as template in the formation of sg-messengerRNA. CoVs have 6 ORFs in their genome and sub-genome. ORF 1 (1a) is the one third of the overall genome. It also encodes 16 non-structural proteins(14). This is not shown in gammacoronavirs because of absence of nsp1. Due to frameshifting polypeptides pp1a and pp1ab are produced in ORF1a and ORF1b. Further modification to these polypeptides also occur by certain methods like papain like protease, chymotrypsin-like protease and by main protease (MPRO) into 16 nsps.

Other ORFs also play their role in spike (S) , envelop, membrane and nucleocapsid (N) protein production because they encodes for the 4 main structural proteins as mentioned above. These are one third of the genome and are found near 3’ terminus. CoVs also encodes for some other structural and accessory proteins. These proteins include HE protein, 4a/b protein and 3a/b protein, all of these are translated from subgenomic RNAs of CoVs. 58% genome sequence alignment is identified as non-structural proteins while 43% shows structural protein coding regions. It is suggested that 54% whole genome level is conserved as nsps and other are structural proteins that are in need of adaptation for the new host. More mutations are found in RNA viruses as compared with the DNA viruses, so the genome of RNA virus are usually less than 10kb in length.(15,16)

But still the genome of CoV is more than 30kb , the largest RNA genome. To maintain such a large genome certain enzymes are required such as 3;-5’ exoribonuclease of nsp14, the function of this enzyme is to provide proofreading to CoV of RTC(17). Certain sequence analysis cleared that 2019-nCoV, posses a typical genome of CoV and contain the following characteristic which shows it belongs to beta-coronavirus, these are as MERS-CoV, bat-SARS-like (SL)-ZC45 and Bat-SL-ZXC21. By studying the phylogenetic tree of 2019-nCoV it is observed that it relates closely with bat-SL-CoV Zxc21 and bat-SL-CoV ZC45 and is in a distant relation with MERS-CoV. (18).

STRUCTURAL AND NON-STRUCTURAL PROTEINS

Most of the non-structural proteins have a role in replication of CoVs. The number of nsps is 16 , nsp1-16, all are involved in replication, function of these proteins is given in the table. There are certain nsps whose function is not clearly understood and work has been conducting on them. For assembly and infection of CoV 4 structural proteins are used. These help the virion to assemble orderly. For the attachment to the host receptors spikes are required on the surface of virus which are produced from the homotrimer proteins (S). Next comes the M protein, it helps in three major domains which as follows, it binds to the nucleocapsid, promote membrane curvature and shapes the virion. For viral pathogenesis E protein plays a vital role. Viral release and assembly is its function. The last structural protein is the N protein that binds the RNA genome of virus through different mechanisms by using both of its domains. It is also believed that this protein helps to connect viral genome to the RTC, and then to cover the encapsidated genome into different virions. Antagonistic role is seen in N proteins , as it works against viral encoded repressor of RNA interference and interference. This antagonistic activity is beneficial in viral replication. (19,20)

INTRODUCTION

Covid-19 has been announced as a pandemic disease by WHO. It has been transmitted over 204 countries and almost more than 16 lac people are infected by this virus. More than one lac deaths has been seen due to this. It is also called as severe acute respiratory syndrome (SARS-CoV-2). It is considered as the third zoonotic coronavirus that has created crisis in human health(21). Its first appearance was observed in Wuhan. Older ones are more affected than the young ones.

Covid-19 doubles in size in 7.4 days and has a specific reproductive numbers as 2.0, mortality rate is ~3.48. It can live for 3 hour in air but for 72 hours on plastic and copper etc. Recent studies reveals that Malayan pangolin are the closest host after bats. Symptoms of Covid-19 include high fever, tiredness and dry cough has been observed with flue. In a study it has been noticed that 20-25% people don’t event have the symptoms even when they are infected. Such people are the major cause of transmission. Most of the people are recovered from this pandemic outbreak. The consequence of this virus is that we are facing shortage of beds and ventilators in our hospitals and it has created a great economic loss and have a negative social impact.

STRUCTURE AND GENOME ORGANIZATION

In the past few years, Covid-19 is the third zoonotic coronavirus that has been emerged to human beings. According to the results of a study being conducted on Covid-19, it is revealed that it resembles almost about 91.02% with the Malayan pangolin, which shows that it is the most closely related host of this virus after Bat-CoV(22). Mutations in amino-acids have been observed in Bat-CoV due to which only four amino acid residues are conserved on it while five amino acids residues are conserved in case of Pangolin-CoV and SARS-CoV-2, that interact with the human ACE2. Covid-19 only differs in a single sequence of putative furin recognition motifs which is not present in both Bat-CoV and Pangolin-CoV. Many genome sequences has been sent to NCBI from different many labs such sequences are as with accession numbers MN988668, MN938384.1 and MT072688 etc.

Covid-19 has single-stranded, positive-sense RNA. It has nucleotide (c-DNA) as 29.811 bp long, which is further broken down to bases in such an order Thymine 9.574, Guanine 5.582, cytosine 5.482 and adenosine 8.903. There are five mutations have been observed in it which are as; T8782C, silent mutation in ORF1a from codons AGT to AGC, T9561C, non-silent mutation in ORF1a from codons TTA to TCA, T29095C, silent mutation in nucleocapsid from codons TTT to TTC, C15607T, silent mutation in ORF1b from codons CTA to TTA, C28144T, non-silent mutation in ORF8b from codons TCA to TTA. (23,24,25)

Various structural and non-structural coding genes has been identified in virion genome. Non-structural protein contains the following ORF8, ORF3a and ORF7a. Structural proteins contain membrane protein M, RNA dependent, RNA polymerase, envelop protein E, spike protein S, 5’ UTR, nucleocapsid protein N and 3’ UTR. In some studies it is seen that mutation in Covid-19 occur in gene level, nsp1 and not in amino-acids.(26,27)

N protein can be proved effective in manufacturing of vaccine because it might have some immune response present in it. This protein is used for the formation of helical structure of virus when this virus is being assembled(28). When SARS-CoV-2 was studies in USA, there were three mutations found in it and all these mutations are present in N protein of the virus. These mutations are 28883G-C, 28881G-A and 28882G-A. Spike glycoprotein mutations have also been observed, most significant mutation found in A-protein D614G(29). This mutation is present in putative S1-S2 junction, near furin region where cleavage of S protein takes place and entry and exit of virus is present. Its structure is represented in the diagram.

SARS-CoV-2 Spike-mediated entrance in the cells may be inhibited by SARS-CoV polyclonal antibodies. S1 domain of the spike protein initially associated with ACE2 receptors. It has been experimentally proved that SARS-CoV-2 utilizes ACE2 to enter target cells(30,31). Also it is used to show affinity towards ACE2 as SARS-CoV, as a result of which spike protein is considered as a vital target. Membrane fusion is on of the mechanism of viral entry to the cell that can cause changes to spike protein which is followed by cathepsin L (CTSL) proteolysis that induces fusion of membrane within endosomes. A study demonstrated that Bat-CoV genome is 96% identical to SARS-CoV-2. (32) 

TRANSMISSION

As we all know that this is a pandemic disease. It has created a great fuss in the human societies. Some people are saying that corona is actually a vaccine for earth as we human are the virus to this planet. But its beyond the limit to talk about this. It is evident that tis virus has started its journey from Wuhan, China. Within a week thousands of people were infected by this virus. As it moves from animals to human and it has ability to transfer itself from touch(33). Human to human contact is the major reason of its spread. According to the director of WHO, about 41% virus is spread through the hospital wastes in Wuhan. 

If we talk about how this virus enters in other countries and territories, it is clear that human sources are the reason behind this(34). Which means that the ones who travelled to china are the carrier of this virus. In the beginning a case was confirmed with corona virus in Thailand, when his travel history was checked, it was noticed that person has travelled to China two weeks ago. It is actually a chain of people who are unintentionally spreading this virus. We can infer from this knowledge that we have to maintain a social distance to avoid this outbreak. This virus can cause moderate to severe respiratory disorders in children. Increased temperature also hinders its fast spread.(36,36)

DIAGNOSIS

Covid-19 is mostly associated with organ failure, asthma and bronchitis etc. and most importantly with respiratory distress syndrome. It is also thought that it invade CNS by using olfactory route. Further molecular investigations has suggested it grow better with human epithelial cells(37). C-reactive protein increased in corona patients and also 83.2% lymphocytopenia is also present in patients. 14 days incubation period is required and suggested for the suspected ones, in which all the symptoms are obvious like high blood pressure, coughing, temperature and many heart problems(38). Many methods are being adopted to diagnose this such as RT-PCR, paper-strip tests (CRISPR). In recent research it is stated that antibody based algorithms will also be used to treat this. Worldwide oseltamivir and oxygen therapy are being used(39).

PREVENTION AND TREATMENT

To restrict viral spread social distancing is an alternative way. Quarantine is another terminology that can be applied in this regard in which ill person are self isolated. Despite of this self-isolation other drugs have also been introduced but this is not the permanent treatment of this Covid-19. Such medicines include antiviral medication of interferon, chloroquine, RNA medicines and many other traditional medicines(41-40).

Doctors are giving malarial, HIV and SWINE flue medicines to the covid-19 patients such as lopinavir and ritonavir respectively two times a day for at least 14 days. For the production of vaccine for SARS-CoV-2 in silico methods will be utilized and for this many targets have been subjected. But now the biggest challenge is to provide the patient with physical ventilators, about 5-6% require these ventilators to survive(42). Telemedical innovations that are present in ICU are much important for Covid-19 patients. To combat this pandemic outbreak rapid diagnostic tests, epidemiologic investigations, vigilance and risk assessment are urgently required by any country. Best way to prevent this novel virus is by maintaining social distance and by avoiding social gatherings and to be self-isolated.