An update of coronavirus disease 2019 (COVID-19): an essen- tial brief for clinicians

1 The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran; afshinzareresearch@gmail.com (AZ), elahe.sadati.1997@gmail.com (SFS), amirhossein.mkh999@gmail.com (AM), nimapakdel@gmail.com (NP), zeinabhamidi74@gmail.com (ZH), neda.baghban@gmail.com (NB), mehrarezoo@gmail.com (AK), inabipour@gmail.com (IN), amintamaddon@yahoo.com (AT) 2 Department of Basic Sciences, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran; s.almasi@bpums.ac.ir (SA) 3 Department of Vaccine Discovery, Auro Vaccines LLC, Pearl River, New York, United State; mbehzadi@aurovaccines.com (MAB) * Correspondences: amintamaddon@yahoo.com (AT) and mbehzadi@aurovaccines.com (MAB); Tel.: +98-773332-8724


Introduction
Reports demonstrate an increasing trend in number of sufferers with fever, cough and deduction in white blood cell count at the end of 2019. "Fever with unknown origin" was chosen for the name of this mysterious illness (1). Based on different documents including clinical characteristics, various analysis of body fluid samplings and radiological RTC then replicates and transcribes a set of nested RNAs that encode accessory and structural proteins (29). By means of endoplasmic reticulum and Golgi's body, recently formed RNAs, nucleocapsid proteins and glycoproteins of the viral coat are collected and form the particles of the virus. Finally, the vesicles comprising the virus combine with the plasma membrane for releasing the virus (30).

Origin of SARS-CoV-2
The primitive surveys demonstrated that the first cases who suffer from the SARS-CoV-2 infection had been reported from local Huanan seafood market (2). Although SARS-CoV-2 was extracted from Huanan seafood market, the origin of the SARS-CoV-2 is under debate because the first SARS-CoV-2 case reported had no connection to the mentioned market (2,7). Moreover, it was discovered that at least two diverse types of the SARS-CoV-2 had been officially reported before COVID-19 and recent surveys presented that the SARS-CoV-2 may imported from other sites to Huanan seafood market (31,32).
Coronaviruses, first found in the 1960s, have been found in birds and mammals, including in camels, bats and rats (33). SARS-CoV-2 has a single-stranded positive-sense RNA genome, which has 74% and 99% similarity with coronavirus from the pangolin (Manis javanica) and (1,(34)(35)(36)(37) the Malayan pangolin (38), respectively. One portion of spear-like protein (S protein) called receptor-binding domain (RBD) in pangolin-CoV has only one different amino acid compared with that of SARS-CoV-2. In addition, the infected pangolins show pathological symptoms as same as humans infected with COVID-19 and the antibodies in their blood are able to react with the spike protein of SARS-CoV-2 (39, 40). Accordingly, the pangolin is considered as one of the probable intermediate hosts between bat and human (38) (Figure 3).
Five out of the six major amino acids in the structure of the RBD of the S-protein from SARS-CoV and SARS-CoV-2 are different, unlike the theories on the laboratory origin of SARS-CoV-2 by manipulating other coronaviruses (48). Different surveys demonstrated that SARS-CoV-2 is an animal virus transmitted to humans through undergoing evolutionary adaptations (36, 49). Analysis of different studies has suggested that SARS-CoV-2 formed by recombining the pangolin-CoV and the bat-CoV-RaTG13-like virus (19,39,50,51). However, more studies are needed for confirming the intermediate hosts of coronaviruses for stopping zoonotic transmission and avoiding the outbreak of such viral infections in feature (52).

Mutant type(s) of SARS-CoV-2
Mutation rate in RNA viruses is extensively high and this rate is associated with virulence modulation and evolvability, behaviors considered to be useful for viral adaptation (53). Early reported studies demonstrated that fast spread of SARS-CoV-2 across world and emergence of the genomes containing new mutation hotspots. Mutation rate of RNA virus plays role in viral adaptation making equilibrium between the integrity of various genetic data and genome variability (54-56).
Biological characterization of viral mutations can supply valuable intuitions for evaluating immune escape, viral drug resistance, immune escape and pathogenesis dependent mechanisms. In addition, it can be vital for drafting new vaccines, antiviral medications and diagnostic tests (57). Therefore, it seems to be necessary to know about mutant types of SARS-CoV-2.
In late January or early February 2020, a mutant type of SARS-CoV-2 was appeared that has D614G replacement in the gene encoding the spike protein. This new strain became dominant type of SARS-CoV-2 in china and spread globally after a short period of time (58). Researches revealed that compared to the initial virus strain, this new class of SARS-CoV-2 has enhanced transmission and infectivity (59, 60). The SARS-CoV-2 virus with the D614G replacement does not make more intense illness or change the efficacy of current laboratory diagnostics, medications, vaccines, or public health preventative criteria (58).
First mutant strain of SARS-CoV-2 United Kingdom was observed in Wales on September and mid-November that was called 501Y lineage (501Y Variant 1). After that, on 14 December 2020, second mutant strain was reported in United Kingdom and named SARS-CoV-2 VOC 202012/01 (Variant of Concern, year 2020, month 12, variant 01or 501Y Variant 2, also named as B.1.1.7). In November/December 2020, this new strain has also become the predominant strain in England (61). This mutant strain includes 23 nucleotide replacements and is not phylogenetically linked to the SARS-CoV-2 virus circulating in the United Kingdom at the time the mutant type was identified. According to surveys, SARS-CoV-2 VOC 202012/01 is more transmittable than early virus strain and it has the capability of reinfection. Although infectivity of this new strain is not obvious (62). Moreover, origination of SARS-CoV-2 VOC 202012/01 is unclear (61).
On 18 December 2020, South African government declared the discovery of a new mutant type of SARS-CoV-2, which quickly spread in three provinces of South Africa. Because of a N501Y mutation, South Africa named this variant 501Y.V2, on the other hand, SARS-CoV-2 VOC 202012/01, which was detected in UK, also has the N501Y mutation, but phylogenetic analysis has demonstrated that 501Y.V2 originating from South Africa is different from mentioned strain in UK (58). Recent surveys revealed that 501Y.V2 is more transmittable in comparison to early strain. In addition, studies showed that this new strain has high affinity to human ACE-2 receptor (63). Furthermore, mutation in this new strain gives ability to stabilize viral bindings with ACE-2 receptor (64-66) as well as ability to escape from human immune system (67). The severity of infection caused by this new viruses is under investigation (67).

Transmission of SARS-CoV-2
Symptomatic patients with coronavirus are the most important distributors; but asymptomatic patients can be one of the important source of viral transmission (68). Transmission through close person-to-person contact, hand shaking, touching contaminated surfaces, contaminated hand contact with eyes, mouth and ears, respiratory droplets and wound, and oral-fecal transmission are known as viral transmission routes (5, 68).
Vertical transmission from mother to infant has been reported (69), but, no intrauterine vertical transmission has been reported for any infant with SARS and MERS in the past (70, 71). In addition, based on all available data, amniotic fluid, umbilical cord blood, neonatal pharyngeal swab and breast milk samples from six newborns born by infected mothers were examined for SARS-CoV-2 and all cases were negative for the virus (71, 72).

Epidemiology of SARS-CoV-2
In a study conducted in China in 2020 on 44,672 patients, it was obtained that the highest number of patients ranged between 50 to 59 years. The lowest number of patients was in the age range of 0 to 9 years. In addition, mentioned survey revealed that occupationally, patients who were retired, workers, and farmers, had the highest incidence of the disease. Additionally, most patients had a mild form of the illness. In addition, the mortality rate was higher in men than women, retirees had the highest mortality rate, age group ≥80 showed the highest mortality rate among all age groups, the mortality rate in patients who had other underlying disease in addition to SARS-CoV2 infection was much higher than in patients without underlying disease. Among patients with underlying problems (cardiovascular disorder, diabetes, chronic respiratory disorder, hypertension and cancer) and patients with cardiovascular problems have the highest mortality rate because of SARS-CoV-2 infection (73).
Data obtained from various studies to date show that in general, different age groups are susceptible to SARS-CoV-2. However, ACE-2 is significantly expressed more in Asian populations than European and American populations, and ACE-2 in male cells is higher compared to the female cells that may partly justify the fact that the incidence of coronavirus pneumonia is higher in men and in Asia. The elderly and people with the disease are more probable to progress a severe and fatal form of the illness. The most common underlying problems that predispose a person to SARS-CoV-2 infection are cardiovascular disorder, hypertension, diabetes, and chronic obstructive pulmonary disease (COPD) (5). Severe complications that this disease can cause in individuals include respiratory distress syndrome (RDS), septic shock and metabolic acidosis that respond poorly to treatment and cause multiple organ failure and coagulation problems (5, 6, 74, 75).

Pathophysiology of SARS-CoV-2
SARS-CoV-2 uses ACE-2 receptors to transport itself to human cells (76, 77). The fluid obtained from bronchoalveolar lavage of a SARS-COV-2 patient demonstrated that SARS-CoV-2 enters in human cells by using the ACE-2 receptors (36). ACE-2 is a surface molecule that is extensively expressed in different regions of human body include lung AT2 cells, upper esophageal epithelial cells, and enterocytes in the ileum and colon so this fact demonstrates a high potent passage in the gastrointestinal and respiratory tract for SARS-CoV-2 virus (76, 77). Different organs of human body can be affected by SARS-CoV-2 infection (15). ACE-2, found in the lower portion of human respiratory tract, is known as a receptor for SARS-CoV-2 and regularizes human-to-human and cross-species viral transmission (43, 78).

Diagnosis of SARS-CoV-2
SARS-COV-2 is diagnosed based on history taking, radiographic images, and detailed laboratory tests (6,79). Current diagnostic tools include nucleic acid testing or virus genome detection (6,79). Samples included nasopharyngeal swabs, sputum, lower respiratory tract secretions, blood, and feces (6,79). The nasopharyngeal swab is the most common example; however, the number of positive cases is less than 50% (6, 79). Repeated diagnostic testing is necessary to increase diagnostic accuracy (6,79). The number of positive cases in bronchoalveolar lavage fluid was high; however, this method is not suitable for most patients due to the increased risk of cross-infection (6, 79).

Clinical manifestations
1 to 14 days and the average of 8 days have been estimated as the incubation period of COVID-19 (80). Fever and cough has been reported as the most common symptoms, while upper respiratory symptoms and gastrointestinal have been rarely reported as the symptoms of this disease (2,4). SARS-CoV-2 infection can cause various sorts of clinical manifestations depends on patient situation (Table 2 and Figure 4). SARS-CoV-2 clinical manifestations related to pregnant women, neonates and children are summarized in Table 3.

Laboratory examinations
Laboratory examinations along with taking history and radiographic analysis are three bases for finding and curing COVID-19 sufferers (125). Studies show different laboratory finding and we gathered different laboratory findings in Table 4.

Histopathological findings
Autopsy of different cases infected by SARS-CoV-2, revealed that this virus can damage various organs; based on this fact, SARS-CoV-2 can cause diverse histopathological views in body organs (126,127). Although, histopathological findings are not certain documents for clinicians to make decision for patients suffering from COVID-19, but they give us remarkable information about pathological changes, pathogenesis of the disease, and the cause of death in COVID-19 cases (125). In Table 5, we discussed histopathologic changes in lung tissues after being infected by SARS-CoV-2.

Radiological findings
Radiological findings in this disease are varied. These findings have been presented in Table 6. It is important to note that radiological diagnostic sensitivity is limited. Therefore, it is necessary to check with clinical signs and diagnosis of RNA virus (5).

Prevention of SARS-CoV-2
The best prevention for the general population at this period of time is to avoid being exposed to the virus (128). Some actions that may deduct amount of exposure of SARS-COV-2 infection include using face masks, utilizing tissue or flexed arm when person coughs or sneezes, washing hands regularly with soap or disinfect hands with sanitizer consists of 60% alcohol at minimum, refusing close contact with suspicious or infected people and keeping a proper distance as much as possible to other people and refraining from touching eyes, nose, and mouth with unwashed hands (129).

Vaccines of SARS-CoV-2
Vaccines are biological drugs that are highly monitored and controlled. Unlike other drugs, which are administered on sick people, vaccines are administered on a huge number of healthy people, so the process of developing them takes a long time and should be strictly observed (130). There are several studies on developing vaccines for COVID-19 and each of them is at different stages. Some of them use messenger RNA methods and some of them use DNA, which is then translated and produces specific immunogenic proteins. Each of them uses different generic platforms, such as inactivated virus, purified recombinant viral proteins with or without adjuvant, replicating and non-replicating viral vectored antigens, antigen-encoding DNA or mRNA (131). Some of the vaccine designs are based on old methods and technologies that have been approved for other vaccines and some of them are built on completely new and novel technologies and have not been tested on large scales vaccinations (131).
Developing vaccines using the traditional ways takes a very long time almost 15 years. The vaccine developing process begins with designing and evaluating vaccine in animal models. Next, several years can take by other steps which are preclinical experiments for designing vaccine production and performing toxicology studies. Then, phase I clinical trials are the new drug on less than 100 people and this phase takes almost 2 years to see if the vaccine candidate is safe enough and has gained preliminary immunogenicity data. If the results are good, the vaccine candidate can move to phase II of clinical trials to test on a few hundred people and it takes another two years. In this phase appropriate dose and optimal vaccine regimen is determined. After this step, if the results are promising, the vaccine development process can move forward to phase III clinical trials in which the vaccine is tested on thousands of individuals to evaluate the effectiveness and safety of vaccine. This phase, which is a very costly process, takes another two years. If the outcome is encouraging and meets the defined end points, an agency like United States Food and Drug Administration (FDA) or the European Medicine Agency approves the biological license application. This licensing process can take approximately two years.
Since the pandemic of SARS-COV-2 has started in December 2019, rapid action and development of vaccine is required. A considerable amount of time is saved by omitting some of the initial steps of exploratory vaccine design due to data from the preclinical development of vaccine candidates for SARS-COV and MERS-COV. In March 2020, the first clinical trial of a vaccine candidate for SARS-COV-2 was started. In this condition, trials are designed such that clinical phases are parallel and have overlaps. Phase I and II are started at the same time and are followed by rapid progression to phase III. Vaccine approval can be expedited through an emergency use authorization. In order to develop SARS-COV-2 vaccine, at least 50% efficacy is required according to FDA guidance (132). In this pandemic, the COVID-19 vaccines should reach at least three goals of reducing severe infection, clinically symptomatic patients and the requirement of their hospitalization, being able to prevent the transmission of the disease between individuals and being capable of generate a strong neutralization response, which prevent the viral protein S from attaching to human cells by binding to it (130).
Currently, there are four main types of COVID-19 vaccines that are going to large scale clinical trials. In Table 7 clinically approved and commercialized vaccines are compared. Below is a description of how each type of vaccine works and induces our body to produce antibody against COVID-19 antigens.

Non-replicating viral vectors vaccines
This type of vaccines is based on SARS-COV proteins expressing on the outer surface of some common viruses like adeno viruses, which are genetically modified. However, the immunity caused by these viruses are neutralized very soon because they are very common and most people come in touch with them. The common cold virus (adenovirus) is weakened and used as a viral vector, which contains the genome of surface S protein of SARS-CoV-2. After inoculating this vaccine, protein is produced and it can attack the corona virus since the immune system of the vaccinated person already has the antibodies specific to this protein. The recombinant adenovirus vector cannot cause a non-stop infection in the body, which received the vaccine because it does not reproduce and it also generates a strong single dose response. This type of vaccine uses type 5 of adenovirus as a vector that delivers the S protein of the coronavirus and teaches the body to detect the S protein corresponding to the coronavirus. This platform is the same as the Ebola virus vaccine (130).

Whole virus vaccines
Live attenuated vaccines are the most potent immunogenic vaccines. They are made by diminishing the pathogenesis of SARS-CoV-2 with genetic engineering as same as BCG and most antiviral vaccines like polio with live strains, anti-measles, and anti-rubella. There is a risk of mutation that can cause these vaccines to be pathogenic again. This type of vaccines provides the vaccinated person with persistent protection as the natural post-infection immunogenicity but it can also cause severe post vaccination reactions. Inactivated viruses are made by inactivating the whole bacteria or virus by heat or formalization. This type has lower side effects but the post vaccine immunity is also less potent than the live attenuated ones (130).

Protein-based vaccines
Subunit vaccines with antigenic fragments are vaccines based on glycoprotein nanoparticles. This vaccine increases the immune response against the SARS-CoV-2 spike proteins by elevating the levels of neutralizing antibodies using Matrix-M adjuvant. The production of antibodies is triggered by the antigen or antigenic fraction in the vaccine. It can have less side effects since some various components like cellular proteins or nucleic acids, which have no special effects on immunogenicity, are eliminated (130).

Nucleic acid (RNA and DNA) vaccines
Nucleic-acid vaccines are the fastest type of vaccines to develop because they do not require fermentation or culture. They are made by inserting mRNA or DNA into some cells and forcing them to make immunogenic viral proteins. Sequencing techniques and reverse genetics have a significant role to shorten the development time of a vaccine during the pandemic (133).
DNA vaccines like flu virus vaccine insert a foreign DNA into the cell's genome host and stimulates cellular immunity. Unlike most vaccines that address the humoral immunity, DNA vaccines stimulate cellular immunity. This type of vaccines has both advantages and disadvantages. Introduction of a live virus strain to the human body can be avoided by this type of vaccines but it also may increase oncogenic risks by inhibiting tumor suppressor genes or incorporating DNA into the host cell genome (134).
RNA vaccines are novel and have not been approved yet but they are candidates for COVID-19. This type of vaccines tries to stimulate the production of antibodies against the viral protein that is found on the surface of the virus spike. These antibodies can neutralize and block the cell infecting proteins in the respiratory tract (133).
Currently more than 50 different vaccine candidates are in trials all over the world. So far, two of them have achieved FDA authorization for public vaccination Pfizer-BioN-Tech and Moderna. There is some information scripted below about these two vaccines (135). Additionally, some remarkable information has been gathered about brand new sort of vaccine for COVID-19.

Treatment of SARS-CoV-2
We discuss treatment of this disease in two categories: supportive therapies and antiviral therapy.

Supportive therapies
Symptomatic and supportive therapy is the mainstay of treatment for SARS-CoV-2 cases (144). Supportive therapy includes different manners which are given to patients with SARS-CoV-2 infection for reaching different curative targets (Table 8). In addition, the plasma of recovering patients with SARS-CoV-2 may be useful for SARS-CoV-2 infection (145).

Antiviral therapy
At the present moment, numerous studies are ongoing to manufacture a vaccine against COVID-19. Though, developing vaccine is a long procedure, and the newly produced vaccine will need several safety assessments (146). Based on estimates, human society should wait at least one year to have an available vaccine against COVID-19 (147). Even after preparing an efficient vaccine, human trials will be a major challenge for scientists (Table 9). Now, COVID-19 is being cured with using broad-spectrum antiviral medicines counting remdesivir and Chinese herbal medicine (148,149).

Management of SARS-CoV-2
At the present time that manufactured vaccines need long time to be available for all people around the world, it is important to manage patients with COVID-19. By knowing this fact, some notes should be considered in managing patients with COVID-19. Care ways of COVID-19 should be established at various levels of management including local, regional, and national levels for suspected or confirmed patients with COVID-19. Patients at the first point of contact within the health system should be screened based on case definitions and an assessment of symptoms, and then, suspected or confirmed cases enter into the pathway. Suspected cases should stay in the care pathway of COVID-19 until their lab tests show negative results. Isolating all suspected and confirmed cases in the shortest possible time, implementing local infection prevention and controlling manners are essential. Patients should be triaged by means of a standardized triage tool to evaluate the intensity of malady. patient's values and priorities as well as local and national policy should be considered if accessible and proper judgments according to clinical situation should be utilized in order to steer handle decisions including admission to hospital and to the intensive care unit (150). For better managing of patients, some valuable data have been collected in Figure5.

Results
This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.

Viral element Function References
Genome (RNA) Transcripting and translating structural and non-structural proteins (15) Nonstructural proteins Block the host innate immune response Envelope Promotes viral assembly and release (15) Spear-like structure Guiding virus to host receptors (152,153) binding to the ACE-2 receptor in the lungs and other tissues

Supportive therapy Target References
Oxygen therapy Control the level of O2 in body [92] Maintenance of electrolyte and water balance Prevent exacerbation of pulmonary edema and decreased oxygen delivery [92] Control of basal acid levels NA [92] Zinc May have antiviral activity [167] Azithromycin has also been found effective in patients with severe respiratory tract infections suffering from viral infection [168,169] Vitamin C (ascorbic acid) enhancing the host immunity [168,170] Corticosteroids Preventing an extended cytokine response accelerate resolution of pulmonary and systemic inflammation in pneumonia [170] Non-steroidal anti-inflammatory drugs (NSAIDs)

Conclusions
At present time, SARS-CoV-2 infection has become world-wide concern and causes many social and economic problems. This fact shows the importance of our approach to this infection. Considering this fact that vaccines for SARS-CoV-2 is not accessible for all people around the world because of limitations in producing, financial problems and political issues, best way to prevent from SARS-CoV-2 infection is minimum contact between people in each community. On the other hand, governments should prepare suitable facilities for scientists and researchers to know more about SARS-CoV-2 and its infection to manufacture proper, economical, accessible and beneficial treatments and vaccines for people all around the planet.