a) Viral features
The RNA viruses, such as the coronaviruses, show a much higher evolutionary speed than DNA viruses, because of the high susceptibility to replication error mediated by RNA polymerase or reverse transcriptase, by the considerable size of the viral population and by the higher replication speed. These rapid evolutionary modifications allow RNA viruses to quickly generate mutations that can allow them to adapt to a new environment, including a new host species. If RNA viruses show an extreme variability, even the most complex DNA viruses still show a certain ability to generate different variants; the latter do not exhibit high mutation rates like RNA viruses since this would affect their functionality, but are subject to variation of specific sites called “hot spots.” Viruses with genetic material composed of a single small DNA strand which, probably, due to a high replication error rate, can reach an evolutionary speed equal to that of RNA viruses are an exception. The quasispecies theory is suitable for explaining the cloud of viral variants that created in the host infected with viruses characterized by high mutation rates; RNA viruses are therefore the main viruses to follow this dynamic, together with some DNA viruses with small genomes .The evolution of coronaviruses and therefore of COVID-19 occurs not only by nucleotide mutations but also by recombination. Although some nucleotide mutations are widely spread in the population, no univocal conclusions have been drawn regarding whether these mutations are responsible for the difference in the virulence of SARS-CoV-2. The number of replacements in the SARS-CoV-2 genome is about 26/year and depending on the size of the genome (29.9 kb), and the virus has an evolutionary rate of about 0.90 × 103 replacements/site/year . In an RNA virus with a genome of ten thousand bases, there will therefore be an average of one mutation for each replicated genome; therefore, in millions of progeny, there will be millions of mutations that create a population of mutants, all very similar but still different. In particular, SARS-CoV-2 such as an RNA virus replicates with a level of fidelity close to the error threshold, and in many cases, even if these viruses are potentially very variable, the small size of their genome limits the number of modifications and the positions of the genome in which they can occur without causing functional loss [13, 14, 17, 29].
In the case of viruses, emergence with pandemic effects is more frequently associated with RNA viruses, such as the Betacoronaviruses, that manifest more than other viruses, probably thanks to their greater genomic flexibility, mutation, recombination, and reassortment phenomena that allow them to overcome species barriers, adapt to different hosts, and give rise to new variants which will have isolated events. On the other hand, the primary host will always be needed to have the infection of the new one, while, in the second case, the pathogen will stabilize in the new population without needing the primary host and an event will develop epidemic that may become endemic . The main obstacle to the emergence of a virus, following a jump in species, is therefore represented by the ability to obtain an efficient transmission of the virus itself between different individuals, from the infected to the healthy, belonging to the new host species. As we reported, a tool for assessing the possibility of emergence of a pathogen is the basic reproductive number (R0), which refers to the number of newly infected generated by an already infected individual in a completely sensitive population [24, 26]. This parameter depends on the number of infected-healthy contacts, the probability of transmission, and the contagiousness of the pathogen. R0 is a function of the balance that is created between the pathogen and the host and, therefore, represents a measure of the degree of adaptation that the pathogen shows in giving an effective infection in each host. For each pathogen, in a specific host of infection and at a precise moment, its R0 can be calculated, which will have high values depending on the adaptation of the virus in it (if R0 shows values of 1, the disease will spread in the population and the pathogen will stabilize in it; furthermore, if the R0 value is close to 1, the infection will have an endemic character, while if the value is greater than 1, the infection will have an epidemic character). For SARS-COVID-19, R0 reveals that for each infection directly generates 2–4 infections. But days of infectivity latency are variable from 3 or 4, and if use R = 4, the number of cases will quadruple [24, 26,27,28].
b) The 2019-nCoV structure
The Coronavirus (CoV), order Nidovirales and family Coronaviridae, are a group of viruses equipped with single-stranded, linear not segmented genome, positive sense RNA (ssRNA +) and provided with envelope, which have a helical symmetry core and a characteristic crown morphology. Until a few years ago, all the viruses belonging to the family Coronaviridae were subdivided in only two genera: Coronavirus and Torovirus, and all the viruses classified in the genus Coronavirus were subdivided in their turn in only three antigenic groups (group I, group II, and group III). The recent discovery of a wide variety of new coronaviruses in different host species has prompted the Coronavirus Study Group of the International Committee for Taxonomy of Viruses (ICTV) to propose the reclassification of the Coronaviridae family into two subfamilies: Coronavirinae and Torovirinae. In particular, the subfamily Coronavirinae now includes three genera: Alphacoronavirus, Betacoronavirus, and Gammacoronavirus [14, 28, 30]. Although we still do not know the direct origin of SARS-CoV-2, most of the CoV belonging to the Sarbecovirus subgenus has been found in bats (such as horseshoe or other species). Therefore, it is possible that these bats could be the origin of SARS-CoV-2 . SARS-CoV-2 has 29 proteins that can be of three types: structural, non-structural, and accessory (Table 3).
SARS-CoV-2 shows projections on its surface (about 20 nm long) formed by the glycoprotein S (spike) that join in groups of three that thus make up a trimer that together with the others form the structures that resemble a crown that surrounds the virion. The main differences of this new Coronavirus compared to SARS virus appear to be localized precisely in this spike protein and that glycoprotein S is the one that determines the specificity of the virus for epithelial cells of the respiratory tract, that is, to be able to bind the ACE2 receptor (angiotensin converting enzyme 2), expressed by the cells of the capillaries of the lungs, and studies have shown that SARS-CoV-2 has a higher affinity for ACE2 than SARS-CoV. Instead, the membrane protein M crosses the lining (envelope) interacting within the virion with the RNA protein and the protein E helps glycoprotein S to attach to the membrane of the target cell. The dimer-hemagglutinin esterase (HE) is a coating protein, smaller than glycoprotein S, and plays an important role during the release phase of the virus replication within the host cell. The envelope, which is the coating of the virus, consists of a membrane that the virus “inherits” from the host cell after infecting it. Coronaviruses are enveloped viruses that carry a genome of positive (+) sense RNA. RNA consists of a single strand of positive RNA of large size (27 to 32 kb in different viruses) and are not known larger RNA viruses. RNA originates seven viral proteins and is associated with protein N (nucleoprotein), which increases their stability (Fig. 2) [30,31,32].
The HCoV infectious cycle is initiated by the binding of virions to cell receptors, which will lead to a conformational change in the S2 subunit in S, thus leading to the condition of the fusion of the viral and cellular plasma membrane. In SARS-CoV-2, protein S cleavage and activation are regulated by transmembrane protease serine 2 (TMPRSS2) to generate fusion-catalyzed and unlocked forms on the cell surface. The gRNA will be the model for the translation of two polyproteins (pp1a and pp1ab), which in turn are broken down to form NSPS. Subsequently, the NSPS induces the reattachment of the cell membrane to form a double-membrane vesicle, where the viral RTCs (replication-transcription complexes) are anchored. The gRNA (full length) is replicated via a-gRNA intermediate, and a nested set of sgRNA species is synthesized by discontinuous transcription and that will encode the structural and accessory viral proteins. Subsequently, the viral products will be assembled in the ER/Golgi intermediate compartment where a smooth-walled vesicle will be created and will be directed towards the plasma membrane to exit via exocytosis and so on; again, another cycle will be created . The measure of nucleotide-level genomic similarity between the coding regions of two genomes (nucleotide identity) of SARS-CoV-2 is 96% Bat CoV RaTG13, 93% Bat CoV RmYN02, 90% Pangolin CoV, 80% SARS-CoV, and 50% MERS-CoV belonging to Merbecovirus . In a large analytical study of 10,022 SARS-CoV-2 genomes collected from 68 countries (mostly from the USA, the UK, Northern Ireland, and Australia), they detected a total of 65,776 variants, showing 5775 distinct variants (Table 4) .
A new variant of the spike protein D614G that increases infectivity and transmissibility has been immersed. Thus, D614G is associated with potentially high viral loads in the patients but not with disease severity. The mutation that modifies the amino acid D614G is passed on as part of a conserved haplotype defined by four mutations that almost always follow together [35, 36]. Recently, an analysis of the number of variants in the open reading in frame 1ab of SARS-CoV-2 genomes, by finally cleaved protein of who showing deletions in non-structural proteins NSP1, NSP4, NSP6, NSP8, RdRp, NSP14 (exon N), endoRNase, and OMT was reported. One of the most important determinants of the pathogenicity of SARS-CoV-2 is the NSP1 protein, and this is quite new, as coronaviruses undergo a moderate rate of mutations, due to NSP4 with proofreading activity. It promotes viral gene expression and immune evasion in part by interfering with interferon-mediated signaling. It probably blocks host translation by interacting with the 40S ribosomal subunit, thus leading to degradation of host mRNA. The data clearly identify the new SARS-CoV-2 viral strain present in individuals from different areas of the world such as Europe and North and South America (Fig. 3) [32,33,34,35,36,37,38,39].
c) Bio-pathogenesis and the immune responses
Coronaviruses infect numerous animal and human hosts and are characterized by a remarkable frequency of recombination, which, together with the high rate of mutation, encourage their adaptation to new hosts and ecological niches . In most patients, the SARS-CoV-2 virus is attacked by the immune system and patients exhibit a wide range of variability in disease severity ranging from asymptomatic to extremely critical. The transmission occurs mainly through secretions and fecal excretions. The upper and lower airways are early involved in SARS-CoV-2 infection, then other systems such as the gastrointestinal, cardiovascular, renal, integumentary systems, with variable clinical manifestations; the central nervous system (CNS) and peripheral nervous system (PNS) can also be involved through synapses, so psychiatric/mental disorders can arise [40,41,42]. Respiratory infective Flügge drops infect the cells of epithelia/endothelia in the upper respiratory tract and progresses to lower regions of the lung and essentially causing respiratory damage of varying severity, as well as pulmonary macrophages (containing angiotensin-converting enzyme 2), neurons, microglia, and other. The life cycle of SARS-CoV-2 when it infects a person follows five steps: attachment, penetration, biosynthesis, maturation, and release. Once the viruses bind to the receptors, they enter the host’s cells [39, 42,43,44,45,46,47]. As we mentioned, protein modeling experiments on virus protein S have suggested that SARS-CoV-2 has affinity with enzyme 2 receptors of human cell angiotensin conversion enzyme 2 (ACE2) to use it as an entry “port” into the cell, and each SARS-CoV-2 virion is approximately 50–200 nm in diameter [31, 40].
The virus escapes the control of the immune system and decreases the number of lymphocytes so that they do not eliminate the virus, and monocytes/macrophages are not regulated and start pumping “cytokine storms” (neutrophil-to-lymphocyte ratio (NLR) is the leading indicator of hypercytokinemia). The virus’ direct pro-inflammatory effect response can cause acute damage to lung tissue and, subsequently, the hyper-inflammation leads to the acute respiratory distress syndrome. The development of SARS-CoV-2 infection towards the lower airways depends on the damage caused to the bronchioles which disrupts the protective coatings of the surfactant released by type II pneumocytes. This leads to alveolar alteration and desquamation of the endothelial cells and therefore impaired gas exchange and subsequent hypoxemia which will lead to acute respiratory distress syndrome (ARDS). But ARDS itself will in turn cause an overactive immune defect (by localizing neutrophils and increasing cytokines), which will lead to an increase in free radicals, cell debris and proteases, and edema resulting from the increase in proteins in the interstitial space, and the consequent vasoconstriction through platelet activation that further alters alveolar gas exchange with severe hypoxemia and tissue hypoxia which will lead to multi organ failure (MOF) [31, 43, 48]. The mechanism of thrombocytopenia in patients then has a multifactorial origin. Furthermore, the combination of viral infection and mechanical ventilation can give additional endothelial damage which will also lead to platelet thrombosis in the lung, thus causing excessive consumption of platelets, and a decrease or capillary morphological alternation can lead to altered platelet defragmentation. But another factor would be that coronaviruses can infect the bone marrow resulting in the activation of an autoimmune response against blood cells [42, 46, 49,50,51]. Histological examination of the lungs of patients who died from ARDS shows diffuse alveolar damage with desquamated pneumocytes II and lymphocytic infiltrates (CD4 and CD8 lymphocytes) mainly located in the interstitial spaces, around the larger bronchioles, and in many cases, there are foci of hemorrhage, and small vessels appearing to contain platelets and small thrombi. The CD4 and CD8 cells (killer lymphocytes) collaborate with B lymphocytes responsible for the production of antibodies. The CD8 destroys the infected cells by cytolysis and necrotizing cytokines instead of the CD4, and releases interferons and interleukins which eliminate the pathogens (beneficial effect) but, on the other hand, may also have harmful effects (immunopathology) [52,53,54].
Human pathogenic coronaviruses (SARS-CoV and SARS-CoV-2) bind to their target cells through ACE2, which is expressed by the epithelial cells of the lung, intestine, kidney, and blood vessels (SARS-CoV-2 binds ACE2 with higher affinity than SARS-CoV).The ACE2 is a monocarboxypeptidase that acts on many molecules within the renin-angiotensin system and other substrates, such as apelin (molecule that regulates blood sugar and increases insulin sensitivity). Expression of ACE2 has been reported in type 2 pneumocytes. Functionally, there are two forms of ACE2: (a) “full-length” ACE2 contains a transmembrane domain capable of attaching its extracellular domain to the plasma membrane. The extracellular domain is a receptor that binds to protein S from both SARS-CoV and SARS-CoV-2. The new coronavirus is evolutionarily related to Bat-SARS, which similarly uses membrane-bound ACE2 as a receptor and (b) the soluble form of ACE2 lacks membrane anchorage and circulates in small quantities in the blood. Some authors suppose that this soluble form can act as an interceptor of SARS-CoV-2 (but also for the other coronaviruses) which by acting can prevent the link between S and ACE2 at the cell surface as shown by some in vitro studies. Furthermore, ACE2 combined with a certain portion of Fc immunoglobulin was neutralizing for SARS-CoV-2 in vitro [40, 42, 50]. Thus, the angiotensin system is involved in viral infection. This activity is linked to the blood coagulation cascade. By blocking the ACE2 receptor, severe vasoconstriction occurs, which can lead to the accumulation of fluids in the lungs. There are clotting factors outside the blood vessels (e.g., von Willebrand factor acts on factor VIII) that, when the damage starts to occur as it also happens with the surrounding vessels, enters the blood and this acts on the clotting factors which can cause phenomena of thrombosis. These form in the lungs and cause damage, acute respiratory distress syndrome, stroke, heart disease, and others. ACE2 is one of the main enzymes in the renin-angiotensin system (RAS) which regulates blood pressure, fluids, electrolyte balance, and systemic vascular resistance [42, 50]. In the lungs, activation of local RAS can influence the pathogenesis of lung damage through multiple mechanisms, such as increased vascular permeability and alterations in alveolar epithelial cells. Activation of pulmonary RAS involves renin, the initial enzyme of the RAS activation cascade; renin cleaves angiotensinogen generating angiotensin I (Ang I, a decapeptide hormone, inactive). ACE converts Ang I to angiotensin II (Ang II, an octapeptide hormone), which is very active, which exerts vasoactive effects by binding to its receptors, type I (AT1) and type II (AT2). There is multiple evidence on the importance of AT2 and ACE2 in the regulation of inflammation, both through activation of the AT2 receptor via angiotensin 2, and through activation of the MAS receptor, via Ang 1–7 produced by ACE2, with a reduction of inflammatory interleukins such as interleukin- 6, Il-5, TNF-alpha, and NF-kB. In addition, some studies show that lung ACE2 deficiency correlates with a worse prognosis since ACE2 and AT2 have a protective action in the lung; ultimately, ACE2 is not only the receptor for the virus but also the protector of lung damage [31, 41, 51]. Some evidence shows that SARS-CoV-2 is able to deregulate the balance of the protective system of the lung formed by the balance between ACE/AngII/AT1 and ACE2/AT2/Ang 1–7 and MAS receptor. Is it a possible explanation that the ACE2 receptor in the severity of the infection induced by SARS-CoV2? Several studies have shown that the host’s cells defend themselves from attack by decreasing the presence of ACE2. The expression of ACE2 is substantially increased in patients with type 1 diabetes or type 2 diabetes, arterial hypertension. Consequently, increased ACE2 expression would facilitate COVID-19 infection. Indeed, diabetes and treatment of hypertension with ACE2 stimulatory drugs are hypothesized to increase the risk of developing severe and fatal pneumonia. Finally, the scholars analyzed the presence of ACE2 also to try to clarify the animal origin of the SARS-CoV-2 coronavirus, initially connected to the bat but later also attributed to the pangolin. The analysis carried out showed a greater similarity between the ACE2 protein of human cells and those of pangolins: a result that supports the hypothesis that the small mammal could have been the original host of the SARS-CoV-2 virus or an intermediate host between bat and man [41, 48, 54] (Fig. 4). In bat-SARS-like CoV, the S1 end has a low similarity degree with the equivalent of SARS-CoV, especially in RBD, while the similarity is high at the level of the S2 end and they are unable to use the ACE2 of humans. These differences imply that bat-SARS-like CoVs and SARS-CoVs recognize different molecules on the surface of the host cell as receptor but exploit the same entry mechanism [55, 56]. The theory of the acquisition by genetic recombination of the binding capacity with human ACE2 by bat-SARS-like CoVs is favored by the high genetic variability characterizing the ACE2 of bats, compared to that of other animals currently recognized as sensitive to SARS-CoV. The high diversity of cell receptors in bats strongly suggests the possibility that there is a species of bat not yet characterized that could act as a reservoir for SARS-CoV-19 or for one of its ancestors, as was hypothesized for SARS-CoV .
d) The immunity over time
The virus can be detected by PCR in whole blood and in stool samples (real-time reverse transcriptase-polymerase chain reaction (RT-PCR)), and the serological blood test looks for antibodies created by the immune system [58, 59]. Some aspects regarding the progression of SARS-CoV-2 still remain uncertain. One of these is the role of cross-immunity with other Coronaviruses (at least the 4 coronaviruses that cause flu in humans) and following the SARS-CoV epidemic and studies led to the belief that cross-immunity between the common cold virus and SARS-CoV-2 is very likely directed against the antigens common to all coronaviruses and not the specific antigens of SARS-CoV-2 [59, 60]. According to what has emerged so far, all patients who have contracted SARS-CoV-2 and are cured have produced antibodies, but there are conflicting theories on their duration (from a few months to the end of the year). If at first it was thought that antibody immunity for SARS-Cov-2 could last at least a year (as ascertained for the SARS-CoV and MERS-CoV coronaviruses), there may be a drop in plasma antibody levels 2–3 months later healing in both patients who were asymptomatic and in those with symptoms. If so, even those who have already contracted the virus once could become vulnerable after a short time and if the infected quickly lose antibodies, they could be negative on serological tests. However, immunity conferred by antibodies is not the only possible one. In several studies, the immunity mediated by specific T lymphocytes, neutralizing antibodies, and other molecules produced by B lymphocytes capable of neutralizing the virus was also highlighted. In addition, some studies reported the possible enhancement of the immune response by probiotic administration [48, 59,60,61,62]. The presence of antibodies, of any type, is not a guarantee of absolute immunity, and the persistence of the antibodies produced remains uncertain [58, 63,64,65,66].
e) Virus influence on human microbiota
Human colonization by microbes in different numbers and compositions begins immediately after birth in any area of the body that comes into contact with the environment, such as the skin; the oral cavity; the rhinopharynx; the lungs; the intestine; the breast; the urogenital system; and, based on recent studies, the placenta and uterine. The microbiota are an organized community of microorganisms, bacteria, fungi, protozoa, which are found in a network of polysaccharides and are attached to a living or inactive surface. An adult human being consists of about 1013 cells colonized by 1014 microbes. These cells maintain the immune homeostasis for defense that is the eubiosis condition. In some cases, as in viral infections, arises a dysbiosis that creates an imbalance not only locally but also to other systems . These pathways of immune communication (crosstalk) are the axes of the microbiota such as gut/lung, gut/brain, and gut/skin [67,68,69,70]. The gut microbiome can be unbalanced (dysbiosis) in many viral inflammation situations such as this in the current SARS-CoV-2 pandemic. Indeed, changes in the fecal microbiome were in fact observed in some infected patients; the genera Lactobacillus, Bifidobacterium, Streptococcus, Clostridium, and Firmicutes were over represented and the genera Coprococcus, Parabacteroides, Roseburia, Faecalibacterium, and Bacteroidetes were less. Firmicutes have a potential influence on intestinal ACE2 expression and Bacteroidetes have shown an inverse correlation with SARS-CoV-2 fecal load and protective effect against inflammation. Firmicutes have a potential influence on intestinal ACE2 expression and Bacteroidetes have shown an inverse correlation with the fecal load of SARS-CoV-2 which has a potential protective role, and this may be a predictor responsible for the course and severity of the infection. In fact, recent studies show that gastrointestinal pathology in patients with infections that are due to rhinoviruses, adenoviruses, infectious mononucleosis viruses, and coronaviruses (such as the new COVID-19 pandemic) can be more frequent and lasting than pulmonary ones. This can be linked precisely to the imbalance of the intestine/lung axis, or from bacterial dysbiosis, or to an ischemic damage of the central nervous system, but also from interleukin-6 which can also be facilitated by nasopharyngeal dysbiosis [71,72,73,74,75]. The most common skin manifestations associated with SARS-CoV-2 infection include a maculo-papular or papulo-vesicular rash, urticaria lesions, and livedo reticularis. The most common areas involved are the trunk, hands, and feet, with little itching experienced and no proven correlation, between skin lesions and SARS-CoV-2 severity and may be a homeostasis alteration of the gut/skin axis with regard to this gastrointestinal involvement, and as it happens for various other respiratory tract infections that can complicate these disorders. In fact, in some pathologies, recent studies report a correlation between intestinal and cutaneous dysbiosis, which has also been demonstrated in some studies through the administration of probiotics. Generally, the probiotics fight the spread of pathogens, strengthen normal flora, and contribute to the creation of a strong immune system, creating a healthy environment that encourages healing and recovery in a natural way, and this may be useful as an adjuvant therapy in SARS-CoV-2 infection. At last, to modulate an active immune response we expect to have a safe and effective vaccine and a specific therapy for COVID-19 as soon as possible [72, 76,77,78,79,80,81].