INTRODUCTION:

Viral infection executes a huge diseases burden on humanity, but our knowledge about pathogenic viruses is rather inadequate. The impression that we know almost everything about viral diseases and that medicine can cope with every possible infection was crushed by the appearance of several lethal viruses that are still out our control. Including HIV, Ebola, Avian influenza and many other (49). Respiratory tract infection are leading cause for hospitalization of infants and young children (84). The most important viral agent in this patient group are respiratory syncytial virus (RSV) and the picornaviruses (25). Other agent that causes various respiratory diseases are influenza and parainfluenza viruses, adenoviruses, coronaviruses, and human mutapneumovirus (5, 27, 28, 36, 46).

An communicable disease outbreak in the China in December 2019 has established with a record number of deaths in China and intermittent spread of infection to other countries (80). In recent update from WHO and other live updates observing institutes, the virus has infected more than 7,50,890 people worldwide with more than 36,405 deaths in different regions and countries. The China, the major hit country, alone recorded more than 3,314 deaths by end of March 2020. This disease proved to be more lethal and showed closeness with Severe Acute Respiratory Syndrome (SARS). In consideration of urgency and to give an identity to current unique symptomic disease, the World Health Organization (WHO) announced a new name for the epidemic disease caused by new corona virus: Corona Virus Disease 2019 (COVID-19) on 11 February 2020.

Coronaviruses (Corona: Crown like shape; Figure 2) (47) were recognized in 1968 totally on the idea of their characteristics morphology as seen in microscope (95). Coronaviruses (CoVs) primarily cause multiple respiratory and internal organ infection in human and animals (71). Although the history of CoVs began in 1940’s (6), the identification of the first human CoVs were reported in 1960’s as causative agent for mild respiratory infection (71). Subsequently named as i) Human CoV 229E (HCoV-229E) and ii) HCoV-OC43 (59, 34, 30) iii) HCoV-Hong Kong University 1(HKU-1) (55) iv) HCoV-NL63 v) Sever Acute Respiratory Syndrome (SARS)-CoV and vi) Middle East Respiratory Syndrome (MERS)-CoV. The first coronavirus are entirely circulated and contributed nearly one third of common cold in human (48). The HKU-1, HCoV-NL63, SARS-CoV, and MERS-CoVs have been related with enteric and neurological diseases (71).

The new virus belonged to corona viruses family and as connected with emergance of latest infection in 2019, it absolutely was quickly named as 2019-nCoV. Later, general terminology was chosen on the premise of associate in nursing analysis of the new coronavirus evolutionary history and also the infectious agent that causes severe acute respiratory syndrome (SARS) and so, new virus has been given the name as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (Figure 1) by the International Committee on Taxonomy of Viruses on 11 February 2020 (8).

Figure 1. A graphical representation of structure of corona virus (SARS-CoV-2). Source: Centers for Disease Control and Prevention – Public Health Image Library. Credit: Alissa Eckert, MS, Dan Higgins, MAM

Figure 2. Representative structure (Electron micrograph of infectious bronchitis virus) of Coronaviruses (crown-like shape). Source: Wikipedia/CDC Credit: CDC/Dr. Fred Murphy

Epidemiology:

According to WHO the emergence by SARS CoV had affected 8422 cases in 32 countries, 916 of which died with fatality rate of 10-15%. (Communicable Disease Surveillance and Response). The major outbreak was happened in Republic of Korea in 2015 (WHO 2014). Newly identified CoVs has incubation period of 2-7 days. (76), (102) An ongoing outbreak of Novel corona virus (COVID-19) pneumonia that hit the city of Wuhan, China in December 2019. Later began to spread to other area of China and also in other countries worldwide (76) and there are no clear symptoms that are observed during the incubation period.

On 11 march 2020, was a declaration of a pandemic (15) (112) and estimate of case fatality rate and infectivity place SARS-CoV-2 and COVID 19 as a significant global treat with WHO risk assessment of very high at a Global level. As of March 18, 2020 SARS-CoV-2, the virus that cause the COVID-19 disease, has infected more than 1,90,000 people globally and caused > 7,800 deaths, with > 90,000 confirmed infection in Western Pacific Region and > 3,300 death and> 74,000 confirmed cases in European region with > 3300 death (15), and > 2344 cases in south Asian region with >72 deaths (106). As of 25 March 2020, According to WHO SARS CoV had affect 4,54,179 people were confirmed in 196 countries, 19,635 of which died (106).

As of April 2, 2020 SARS CoV-2, the virus that cause the COVID 19 disease, has infected more than 8,96,450 people globally and caused more than 45,526 deaths, of which in Western Pacific Region more than 107626 confirmed cases and more than 3723 deaths. In European Region more than 503006 confirmed cases and more than 33604 peoples are died. In South East Asian Region more than 5324 confirmed cases and more than 216 peoples are died. In the Region of America had more than 216912 confirmed cases of which 4565 peoples are died (107).

On 22 May 2020 the number of infected people due to the coronavirus more than 4,993,470 people globally and caused more than 3,27, 738 deaths (108). Up to the date 14 June 2020, globally the Corona infected patients more than figure 7,690,708 and caused more than 4,27,630 deaths (109). And day by day the confirmed cases of infection increases very rapidly.

Taxonomy:

Coronaviruses are members of 2 subfamilies of Coronavirinae and Torovirinae within the family of Coronaviridae, which successively comprise the order Nidovirales (21). The Coronavirinae taxonomic group is additional classified into four main genera: alpha-coronavirus (α), beta-coronavirus (β), gamma-coronavirus (γ) and delta-coronavirus (δ) supported the international Committee for Toxonomy of Virus (fig 1). HCoV-229E and HCoV-NL6 belong to α-coronavirus, HCoV-HKU1, SARS-CoV, MERS-CoV, and HCoV-OC43 are β-coronaviruses, which they every infect entirely mammals. γ-Coronavirus and δ-coronavirus infect birds, but variety of them will even infect mammals (111). supported current sequence databases, its been discovered that each one humanCoVs have animal origins; SARS-CoV, MERS-CoV, HCoV-NL63, and HCoV-229E are thought of to possess have originated in bats; HCoV-OC43 and HKU1N are likely originated from rodents (92,26).

Types of coronaviruses:

Coronaviruses infect a wide variety of animal species ranging from fowl to humans. The group comprises 11 recognized viruses, and five more that are tentatively included (Table 1). Some members, notably infectious bronchitis virus (IBV) and mouse hepatitis virus (MHV), can be distinguished into several serotypes and others, e.g., human coronavirus (HCV) and human enteric coronavirus (HECV), are presently considered as distinct, although they may be closely related.

Table 1. Growth of coronavirus in tissue culture. Unless otherwise stated the data are to be found in Robb and Bond (1979a), Tyrrell et al. (1978), and McIntosh (1974)

Sr. No.	Types of virus	Host	Tissue culture	Reference
1	IBV (infectious bronchitis virus)	Chicken	Embryonated, de-embryonated eggs Chicken explants	11 18
2	CCV (canine coronavirus)	Dog	1°/2° dog kidney cells	29
3	FIPV (feline infectious peritonitis virus)	Cat	1 ° kitten peritoneal Cells	23
4	HCV (human coronavirus)^a	Human	Human (embryo) trachea tissue	58
5	MHV (mouse hepatitis virus)	Mouse	1°/2° mouse (embryo) Cells	20
6	BCV (bovine coronavirus) NCDCV (neonatal calf diarrheacoronavirus)	Cattle	1⁰bovin (embryo) cell	89
7	TGEV (transmissble gastro-enteritis virus) HEV (hemagglutinating encephalomyelitis virus)	Pig	Porcine organ culture 1⁰/2⁰ porcine (embryo) cells	9 71
8	RCV (rat coronavirus)^a SDA V (sialodacryadentitisvirus)^a	Rat	1⁰ rat cells 1⁰ rat cells	88
9	TCV (turkey coronavirus)	Turkey		88
	Unclassified
10	RTCV (runde tick coronavirus)	Tick, seabird		88
11	FECV (foal enteritis coronavirus)	Horse		7
12	HECV (human enteric coronavirus)^a	Human	Organ culture of human intestine	54
13	PCV (parrot coronavirus)	Parrot	Embryonated eggs	35
14	Coronavirus isolates SD/SK	Mouse, human	l7Cl-l, DBT	31
15	Porcine isolates (e.g., CV 777)	Pig		69

Note :^aIt is not yet clear how distinct these viruses should be considered

Fig. 1. Schematic representation of the taxonomy of Coronaviridae (according to the International Committee on Taxonomy of Viruses). The six human coronaviruses belong to the Alpha- and Beta-coronaviruses genuses, respectively.

Structure:

Progress in coronavirology is illustrated by the number of workshops convened and reviews written. International meetings have been held in Germany (1980), the Netherlands (1983) and the U.S.A. (1986), and the Fourth Coronavirus Symposium will be organized by one of us (D. C.) in Cambridge, U.K. in July 1989 (101). In addition, reviews have appeared which highlighted particularly interesting characteristics of the family, e.g. the replication strategy (53) and the glycoproteins (90).

As the last general accounts were published some years ago (86,91) an update is timely. Coronavirus virions are spherical, engulf virus particles, ranging from 80 to 160 nm in diameter (60). The structure identification of SARS-CoV-2 virus is important towards understanding its historical likeness with different viruses which of corona virus family, incidence and probable mode of infection, mechanism of entry at tissue website of infection and multiplication method within the infected cells. The etiologic agent virus liable for COVID-19 infection has been known as a coronavirus by exploitation electron microscopic imaging examination of a thin-section virus isolate (Figure 2). The Scanning microscopy pictures of SARS-CoV-2 virus showed that virions are spherical in form with diameter of 70-80 nm. The virions are composed of a spiral like nucleocapsid at interval associate degree envelope with surface projections (Figure 2). The transmission microscopic images of the SARS CoV- 2 a pair of show the virus internal structure as double walled with lobe like surface comes, the cytoplasm containing cavum like spherical structures (Figure 5). Further, because the SARS-CoV-2 belong to coronavirus family with SARS likeness, it is expected to be carrying different distinct ultrastructural features of coronaviruses such as: double-membrane vesicles, nucleocapsid inclusions, and huge granular areas of cytoplasm (10). Additionally, becauseit is that the case for different coronaviruses, the SARS-CoV-2 virus particles halficals protoplasm part ought to be containing infective agent as well as the RNA as genetic material (10). Recently, it is unconcealed that, the SARS-CoV-2 could be a positive-strand RNA virus infecting severe respiratory syndrome in human.

Figure 2. The coronavirus SARS-CoV-2, shown in a scanning electron- microscope image. Image reproduced as provided for public domain use by National Institute of Allergy and Infectious Disease, US. Credit: NIAID-RML/de Wit/Fischer

LIFE CYCLE OF SARS‐COV‐2 VIRUS AND INFECTION:

The 2019 novel coronavirus is zoonotic in origin J. Millan 2020 and have a multiplication cycle in respective animal, though not clear as of now. In human beings the virus primarily infects the cells of respiratory system, the covering cells of sacs in the lungs. Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for SARS coronavirus (74) The virus binds to these receptors and make entry in the cell where it duplicates its genome materials and synthesize different required proteins using the cellular machinery, and then buds out new virions from cell surface (118). The researchers at University of Hong Kong has grown the virus in cell culture and microscopic image of the cells infected with SARS-CoV-2 show a large number of virus release from the surface of cell (figure 3) (52).

Figure 3. A microscope image of a cell infected with the novel coronavirus, grown in culture at the University of Hong Kong. Multiple virus particles are being released from the cell surface. Credit: John Nicholls, Leo Poon and Malik Peiris - The University of Hong Kong (52).

The similar unharness of virus had been discovered with SARS vrius infected cells. In earlier report with SARS, the ultrastructural examination of a bronchiolar lavage specimen from a SARS patient had shown varied coronavirus-infected cells, with features similar to those in infected culture cells (10). The virus multiply within the infected cells, victimisation the cellular machinary of cell for infective agent RNA synthesis additionally as macromalecule synthesis needed for brand new virus generation. The entire mechanism for the SARS-CoV-2 is nonetheless to be understood, the preliminary knowledge with alternative coronaviruses like SARS would be handy in making the map for mechanism of novel virus multiplication.

Sign and Symptoms of patient infected with COVID19:

The symptoms of COVID-19 are mainly for respiratory disorders and similar to severe acute respiratory symptoms. The common signs of infection observed in COVID-19 patients include respiratory symptoms, snizzing, fever, cough, shortness of breath and other breathing difficulties. In case of severely infected patients, infection can cause pneumonia, severe acute respiratory syndrome (SARS), kidney failure and even death in many cases. There are many people who had high viral load but do not develop COVID-19 symptoms like coughing or snizzing (116). Such asymptomatic people serve hidden carriers of virus and may further contribute in enhanced transmission of virus to other peoples (117).

In clinical investigation study of COVID-19 infected patients from different regions of China by the Guan et. al. (102) showed a varied pattern of the illness. The study reports that the median age of the infected patients studied was 47 years indicating the presence of infection in people of all ages. Further, out of total patient studied, 41.9% were female, a point indicating towards gender neutrality of infections spreading in different patients. The report states that the primary composite end point occurred in 6% patients. The patients from outside of the Wuhan city either had a contact with the residents of Wuhan city at some point or had visited the city recently (102). In the patients admitted for COVID-19, very few (only 1.9%) of the patients had a history of direct contact with wildlife; indicating the support for human-to-human transmission of SARS-CoV-2 virus (43).

The most of the patients had a standard symptom of fever and cough. Several of the patients typically conferred while not fever, however, developed it in due course of infection. Majoryofpatients developed fever (43.8% on admission and 88.7% through out hospitalization) and nearly two-third of patients hadcough (67.8%) as common symptoms. The biopsy test showed Lymphocytopenia (it is the blood disorder in which abnormally low level of lymphocytes within theblood) in majority of the patients (83.2% of the patients) onadmission to the hospitals. Looseness of bowels (diarrhea) was not common in most ofthe cases as solely 3.8% of patients had diarrhea. The patientsdeveloped full symptoms of the COVID-19 in 2 to 7 days (115) i.e.the median incubation period of infection development was 4days with interquartile range of 2 to 7 days in all told patients (102).

Detection of Novel corona SARS-CoV-2 (Ran Lui):

Usually, this newly identified coronavirus has an incubation period of 2-7days, (102,76) and there are no obvious symptoms that are observed during the incubation period. However, the virus is contagious and can spread from an infected person to a non-infected person during the incubation period. Nucleic acid (NA) testing is reported to beessential for the confirmation of SARS-CoV-2 infection. Therefore, in this report, we have summarized some newly developed and promising methods to detect SARS-CoV-2 (Figure 4), in order to facilitate the development of novel approaches for early diagnosis.

1 Whole genome sequencing:

Whole-genome sequencing is one of the most comprehensive approaches for the identification of the viral NAs. The first complete SARS-CoV-2 genome sequence was uploaded on GenBank (accession number MN908947) by Zhang et al. on January 5, 2020. Five typical open reading frames (ORFs) were identified in the SARS-CoV-2 genome: ORF1ab polyprotein (7096 amino acids), spike glycoprotein (1273 amino acids), envelope protein (75 amino acids), membrane protein (222 amino acids), and nucleocapsiein (419 amino acids). As of February 6, 2020, 19 other genome sequences of SARS-CoV-2 obtained by using Illumina or Nanopore platforms were also uploaded on GenBank by researchers in China, (37), (51) the United States, and Australia. As whole-genome sequencing is relatively expensive, time consuming, and complicated, it is unsuited for urgent and large-scale testing.

2 Real time (RT-PCR):

Real-time reverse transcription polymerase chain reaction (RT-PCR) is the most popular testing method for the detection of SARS-CoV-2. In this method, the SARS-CoV- 2 RNA is first reverse transcribed into cDNA, and specific gene fragments within the cDNA are amplified using target specific primers. The fluorescence signal, which represents the copy number of the target sequence, can be easily detected during the amplification process. The U.S. Centers for Disease Control and Prevention (CDC) recommended to use 2019-nCoV_N1, 2019-nCoV_N2, and 2019-nCoV_N3 primers and probes that target the nucleocapsid gene (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panelprimer-probes.html)whereas the Chinese CDC recommended to use the ORF1ab and nucleocapsid primers as the targets for the detection of SARS-CoV-2. Real-time RT-PCR is specific, rapid, and economical; however, by using this approach we cannot precisely analyze the NA sequence of the amplified gene fragments, and thus all the target fragments that are successfully amplified are considered to be positive. In addition to these limitations, sample preparation, laboratory conditions, and technical errors can lead to false-negative results. Also, several reports have revealed false-negative results obtained by using real-time RT-PCR kits. Besides, testing for viral NAs using this method must be carried out only by experienced technicians in qualified laboratories, making its practical applications by clinicians limited.

3 Nanopore target sequencing:

In the laboratory Ran Liu et. al and their colleagues combined the benefits of target amplification and real-time nanopore sequencing to develop a completely unique method, which is designated as nanopore target sequencing (NTS; unpublished data) (76).The results of his experiment revealed the detailed NA sequences and may thus reflect whether the virulence genes were mutated during the spread of the virus, thereby providing substantial information for further epidemiological analysis.

4 Antibody based immunoassay technique:

Antibody assays also can be used, using a blood serum sample, with results within a some days (57). Although some test strips are developed using the antibody-based immunoassay technique, no reports of the identification of SARS-CoV-2- specific antibodies are published yet (76).

5 The clustered regularly interspaced short palindromic repeats (CRISPR):

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is employed in another promising method for NA detection. Cas13a (previously called as C2c2) (80) may be effectively reprogrammed with CRISPR RNAs to provide a platform for specific RNA sensing. Upon recognition of its RNA target, activated Cas13a engages in “collateral” cleavage of the nearby nontargeted RNAs (66). Zhang and Collins’ laboratories have collaborated to develop the SHERLOCK method that supported the CRISPR/Cas13a system (41). This method uses the reverse transcriptase recombinase polymerase amplification not ti mention T7 transcription to amplify the target RNA fragments of Zika virus or Dengue virus. These target RNA sequences further activate Cas13a, and also the activated Cas13a then cleaves the reporter RNA (quenched fluorescent RNA) with allowing for real-time detection of the target RNA. Therefore, this SHERLOCK method could theoretically be developed as a rapid detection method for SARS-CoV-2.

All the above methods may be effectively deployed for the diagnosis of COVID-19 in neumorous settings. As an exampl, real-time RT-PCR remains the foremost still the most widely used detection method in large hospitals, whereas antibody-based immunoassay techniques, paper-based biomolecular sensors, and also the CRISPR-Cas system-based methods (e.g., SHERLOCK) are still expected to be further developed as large-scale screening methods which will even be utilized in the house setting. Moreover, NTS and whole-genome sequencing are the most comprehensive detection methods. Also, NTS combines sensitivity, comprehensiveness, rapid detection, and low cost, making it the foremost suitable method for the detection of suspected infection that cannot be diagnosed effectively by other methods.

Other heterocyclic antivirals such as Umifenovir (Arbidol), Galidesivir and Garunavir used as antiviral for HIV/H1N1/H1N5/SARS are actively pursued for evaluation for SARSCoV- 2. Oseltamivir (brand name Tamiflu), a most widely used neuraminidase inhibitor for treatment of influenza has also been recommended for COVID-19 symptoms (32). In addition to heterocycles, angiotensin-converting enzyme 2 (ACE2)-based peptide, 3CLpro inhibitor (3CLpro-1) and vinylsulfone protease inhibitor, may presumably show and can be evaluated for potential antiviral activity against SARS-CoV-2 (44).

Herbal treatment for management of Coronavirus disease (Sai Sailesh Kumar Goothy):

The leaves of tulsi are consumable and is being used to normalize the kapha and vata (85). Tulsi is being used in the management of pain, diarrhea, cough and fever which are the common symptoms of COVID-19. (61,68) Tulsi have been used in the management of fever ranging from normal fever to malaria fever. (63) The leaves of tulsi, in addition with cow ghee, were described as the best medicine for pneumonia. There exists a strong scientific evidence for the antiviral effects of Tulsi (85,61,20,11,68,62). Tulsi has been proven to be effective in inhibiting several deadly virus like Newcastle Disease virus, Vaccinia virus and Infectious Bursal Disease virus (73). Clinical trials conducted earlier in India, where the extract of tulsi leaves was administered for patients with viral hepatitis and encephalitis. Interestingly there was an increase in the survivaland symptomatic improvement in the tulsi group when compared with controls. (75,19) Another study proved improvement in respiratory parameters and relief from symptoms of asthma with three days of consumption of tulsi (82). The striking feature in using Tulsi is that it not only restore physiological functions but also restores the psychological functions. Phenolic compounds and antioxidant properties of Tulsi were reported to contribute its therapeutic effects. Tulsi consumption increases the anti-oxidant molecules and enzymes in the body and protects the cells and its membrane from being damaged by the toxic substances (84). Tulsi boosts the immunity of the body and helps to defense the threatening virus and bacteria (62). Improvement in humoral and cellular immunity was observed in animal studies after treatment with tulsi oil. The possible mechanism for improving immunity is a modulation of the GABA pathway. Due to its multi-modal therapeutic effects, we hypothesize that tulsi may be effective in the prevention and management of COVID-19. Though the existing literature supports the management of symptoms of COVID-19 using tulsi, a lack of standard formulation limits its use. This is the need of time to start with translational research to provide scientific evidence for the efficacy and to establish the standard formulation of tulsi in the management of COVID-19.

Prevention: (110,113)

The best way to prevent the spread of this virus is to avoid or limit contact with people that are showing symptoms of the virus and have traveled to china in the past months. The next best thing you can do is practice good hygine to prevent bacteria and viruses from spreading.

Isolation of confirmed or suspected cases with mild illness at home is recommended. The ventilation at home should be good with sunlight to allow for destruction of virus. Patients should be asked to wear a simple surgical mask and practice cough hygiene. Caregivers should be asked to wear a surgical mask when in the same room as patient and use hand hygiene every 15–20 min (94).

· Wash your hands frequently for at least 20 sec at a time with warm water and soap.

· Don’t touch your face, eyes, nose or mouth when your hands are daily.

· Won’t go out if you’re feeling sick or have any cold or flu symptoms.

· Cover your mouth with the inside of your elbow whenever you sneeze or cough; Throw away any tissues you use to blow your nose sneeze right away.

· Keep any objects you touch a lot clean, use disinfectants on objects like phones.

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Received on 24.06.2020 Revised on 20.07.2020

Asian J. Pharm. Res. 2020; 10(4):275-285.

DOI: 10.5958/2231-5691.2020.00048.9