Nagaraja Sree Harsha1,2, Juan Rivas-Santisteban3, Roopashree T Satish4, G S Kumar5
1Department of Pharmacy Practice, College of Clinical Pharmacy, King Faisal University,
Al-Ahsa, Saudi Arabia.
2Department of Pharmaceutics, Vidya Siri College of Pharmacy, Off Sarjapura Road,
Bangalore -560 035, Karnataka, India.
3Department of Biotechnology and Biomedicine, Faculty of Biology, Environmental Sciences.
4Chemistry, University of Alcalá, Madrid-Barcelona Road, Km. 33.6, Edificio De Ciencias Calle 24 28805,
Alcalá de Henares, Madrid, Spain.
5Department of Pharmacognosy, Government College of Pharmacy, Bengaluru, India.
Viruses are change-prone entities often used as models to study evolutionary mechanisms. SARS-CoV-2 has shown a rapid adaptation to its new host, the human. In addition, it is now widely considered to be the most infectious RNA viral particle in humans, due to both efficiency in transmission mechanisms and exposure. Despite the large number of published articles that shed light on this virus, there is no analysis of the quantitative change in the ultrastructure of SARSCoV-2, although it is a useful tool for understanding the evolutionary pattern. To avoid the emergence of at least three completely different viruses, it should appear that an effective vaccine contains the diversification of the SARS-CoV-2 evolutionary tree. In addition, a greater understanding of the physicochemical characteristics acquired by each of the viral haplotypes is required. In the meantime, the following discussion is offered to update developments in this research topic and to relate them so we can answer questions that, until now, have not been addressed.
Coronavirus disease is caused by SARS-CoV-2 (severe acute respiratory syndrome, second coronavirus), a single-stranded, positive-polarity ssRNA virus from Nidovirales order corresponding to Orthocoronavirinae subfamily, which is included within the family of Coronaviridae. This subfamily consists of four genus, according to their genome architecture: Alphacoronavirus, Gammacoronavirus, Deltacoronavirus and Betacoronavirus, the last one including SARS-CoV-21.
To date, SARS-CoV-2 associated disease has caused more than 5.5 million known infections and 346,964 deaths. It is estimated that the number of infected people could be 10 times higher, value that is inferred from the absence of effective mass screening methods to detect the disease and prior knowledge on infectious viral diseases2. The pathology that the patient develops consists of pneumonia, which may be complicated or not according to different elements, such as age or the state of the immune response, while other influent factors remain unknown. It produces flu-like clinical symptoms, including fever, dry cough, dyspnoea, myalgia and fatigue. The transmission of viral particles is widely regarded as analogous to any other pathogenic virus. It would occur through Flügge droplets emitted by a carrier, which when released by speaking, sneezing, coughing, or breathing; the viral particles pass directly to another person through inhalation, or just remaining in the fomites surface3,4. However, there are some problems with this explanation. To date, this it is the most infectious viral disease that humans have known in modern times. Furthermore, there is enough evidence to question that the high rate of infectivity is due only to a high spread and exposure of humans to the virus5. This review proposes a cross-sectional and integrated view between the current disease situation and the characteristic evolutionary pattern shown by SARS-CoV-2, to extract information on the conditions under which this virus has emerged in humans, and potentially prevent a similar pandemic in the future.
The basis of the infectivity effectiveness of this new virus necessarily lies in its ultrastructure, which is a direct expression of its genetic code6. Knowing how the genome of these viruses differs between members of their family, and between different SARS-CoV-2 haplotypes, we can induce what will be the ultrastructure of a new strain, and its effectiveness in infecting different strata of the human population7.
The morphological characteristics of the SARS-CoV-2 strains are, in fact, similar to those of other members of the Coronaviridae family. The most related coronavirus that has affected humans is SARS-CoV-18, so determining whether there are significant differences in its structure may reveal a rapid evolutive pattern. In a 2004 study, the ultrastructure of SARS-CoV-1 was addressed6. The sprouting particles were formed by juxtaposition of viral nucleocapsids in RER and Golgi membranes, averaging 78nm in diameter. Multi- nucleated syncytial cells were often observed in tissues infected with that virus. Back then, similarities were also found among other Coronaviridae members. These sameness included peculiarities of the cytoplasm in infected cells (large granular areas, instead of organelles there was RNA and viral proteins. The “granular area” origins are not clear, but they are closely associated to viral gene expression, for being near ribosomes), tubular structures within vesicles harbouring virions and tubuloreticular cytoplasmic structures (found in other viral infections).
Structurally, new coronaviruses are spherical viruses with 100-160nm in diameter. They are slightly larger than previous SARS coronavirus, although still within the expected size distribution for Orthocoronavirinae (50-200nm).
These particles maintain a lipid bilayer envelope and containing positively polarized ssRNA between 26 and 32 kb in length. The SARS-CoV-2 virus genome encodes four structural proteins: protein S (spike protein), protein E (envelope), protein M (membrane) and protein N (nucleocapsid). Protein N remains inside of the virion; associated with viral RNA, while the other three proteins are associated with the viral envelope. Protein S forms structures that protrude from the envelope of the virus. Also, Protein S contains the receptor- binding domain of the cells it infects and is; therefore, the tropism-determining viral protein. In addition, the protein has the biological task of fusion the viral lipid layer with the cellular membrane, permitting the viral genome to be released inside the cell that it is going to infect9. Further research on this area is required to monitor unknown changes and establish a relationship between these and the three main haplotypes of SARS-CoV-2.
It is well known that viruses need certain physical conditions for optimal propagation and replication of their genetic content in the host cell4,6,10. However, that not only concerns the conditions inside the cell or its environment, they also affect the physic or chemical conditions of the fomite and the environment that surrounds it.
This have major relevance, since in high exposure and global diseases, such as SARS-CoV-2, confinement is a mandatory way to contain the spread. It is in these passive vectors that the viral particles remains a certain time (determined by the nature of the fomite), which continues to infect people in a confined state11. The incubation period of SARS-CoV-2 infection generally ranges from 3 to 7 days, it rarely goes up to 14 days, and in some cases the 25-day span could be dismissed as extremely rare. This means that during incubation the individuals could be exposed to a greater amount of inoculum, which allows exceeding the threshold of viral load that our immune system can deal with, causing symptoms of the disease4,8.
Therefore, the optimal conditions for viral particles to propagate are not those in which the virus replicates (there is a prominent replicative rate in the nasopharyngeal mucosa, as well as in the lungs); if not those that preserve the structural integrity of the virion from the external environment physicochemical features without degrading its gene content too much11.
The permanence of viable SARS-CoV-2 particles on copper, cardboard, stainless steel, and plastic surfaces it was 4, 24, 48 and 72 hours, respectively at 21-23ºC and with 40% HR. In another study, at 22ºC and 60% humidity, they stopped detecting the virus after 3 hours on the surface paper (print or tissue paper), after 1 to 2 days on wood, clothing or glass and more than 4 days on stainless steel, plastic, money bills and surgical masks. In addition, SARS-CoV-2 viability has recently been demonstrated under experimental conditions for three hours in aerosols, with an average half-life of 1.1 hours12.
From these results it follows that summer periods, in extremely hot countries, can therefore reduce the rate of infection in most viral diseases. Not only for viral particle thermodynamics, but also for many other issues related to social activity, the usual ventilation of closed rooms, and the dispersion of Flügge drops4,13.
However, this is not clear to occur with the new global disease. The infection rate was high on the first stages in hot and cold countries, without significant differences14. This fact can be interpreted in two ways: either the molecular structure of this virus is more resilient to inclemency of its nearby environment on the surface of the fomite, or the rapid evolution of the virus is mediated by a temperature-induced mutagenesis, which leads to a more efficient transmission. That temperature influences the evolutionary rate of viruses is well known. This would allow the development of new viral particles with partially degraded RNA content, generating a much more varied gene polymorphism, which due to stochastic, sometimes generates quasi-species with greater competitiveness in different kind of environments.
This hypothesis would explain the fact that there are three main clades of SARS- CoV-2 in such a short time: V-clade, G-clade and S-clade; all with their own characteristics linked to a geographical region, the climate and its population11.
In a study published on March 30, one plausible haplotype map was published7. The map contained 160 selected SARS-CoV-2 sequences extracted from a database that GISAID published earlier in the same month, with a total of 253 partial and complete sequences. In addition, to root the phylogenetic network, the BatCoVRaTG13 bat coronavirus genome, isolated in Yunnan province, was used. This tree involves an informative photograph about the status of the outbreak in the initial stages of the pandemic. In addition, this fact stems directly from subsequent intercontinental migration and interaction.
Although these three major clades are closely linked to the geographical origin in the evolution of the virus11, they should not suppose notable structural differences between the strains. However, there are somewhat earlier classifications based on population genetics analyses of 103 SARS-CoV-2 complete genomes: the S and L strains15. Strain L is considered more harmful and virulent than S, so it seems that the interaction with the human system has favoured an increase in virulence in L strain. From this fact, it is inferred that protein S is plesiomorphic. In addition, it is quite likely that these changes are due to selective pressures and not by recombination. A noticeable difference between S strain and the rest of Coronaviridae members is that protein S of the new coronavirus is longer than its bat counterparts, but also that the S proteins of SARS-CoV-1 and MERS-CoV.
Another question to consider is, was SARS-CoV-2 actually introduced to humans recently? A fact that may answer this statement is the identity analysis among the isolates of the first seven patients detected in Wuhan. The results were clear; they had 99% homology to each other. After performing the phylogenetic analysis of these sequences, observed high homology with viruses of the genus Betacoronavirus, specifically 88% of identity with two isolated bat coronaviruses in 2018. These sequences showed; however, a lower sequence homology with the SARS virus (79%) and the MERS virus (50%). This difference from SARS- CoV was considered enough to classify this pathogen (SARS-CoV-2) as a new member of the genus Betacoronavirus. On the other hand, SARS-CoV-1 penetrates the cell using as receptor the angiotensin-converting enzyme 2 (ACE- 2). Although the structure of the glycoprotein-made envelope of SARS-CoV-2 is slightly different from that of the SARS-CoV-1, ACE-2 has been shown in vitro to remain a valid receptor for SARS-CoV-212.
Perhaps the most striking structural change present in SARS-CoV-2 is a deletion of 8 amino acids found in a non-structural protein, virulence factor 1. This study16 claims that the pp1ab protein is different from other Betacoronavirus subfamily members, as it features a 42 aa signature that SARS-CoV-2 exhibits, although clade 2 members of the Sarbecovirus have traces of this signature, perhaps delaying a convergent evolution, or maybe the evolutive branch origin of SARS-CoV-2.
Like other RNA viruses, SARS-CoV-2 viruses emerge as a diverse (yet related) population of viruses or quasispecies (13). It has recently been discovered that the new coronavirus has relatively abundant regions that suffer from recurrent mutations. Together they total 198 different positions, of which 80% are consistent missense mutations (17). This has been interpreted as an evolutionary adaptation to the new host, the human (18). The fact that these specific regions concentrate the majority of mutations will allow a faster advance in the SARS- CoV-2 vaccine research.
The fact that this virus has degenerated both from the first diagnosis and to form three haplotypes (with subtypes in each country) raises another question for us: is the SARS-CoV-2 evolutionary rate higher than normal, and could this be dangerous for humans? To try to answer this question, the nonsynonymous mutations in the ssRNA chain need be related to the ultrastructural changes that the virus has undergone since the human disease outbreak appeared. This would demonstrate a clear adaptation to the human immune system in a very short period. This way, we normalize all evolutionary change produced by genetic drift. Thus, we are left only with relevant changes regarding human adaptation of SARS-CoV-2.
Could this be dangerous for the development of a stable vaccine? Actually, we have many vaccines that require annual or bi-annual inoculation. This procedure helps updating the antibodies that memory B-lymphocytes express. However, there may be a casuistry that global exposure to SARS-CoV-2, together with an efficient and fast evolutionary pattern; generate at least three different viruses before a traditional vaccine arrives (19). The real danger is not the high mutagenesis ratio per se, but evolutionary divergence and coevolution exhibited by SARS-CoV-2 (20).
Currently, the disease has been contained in some countries, which correlate with the first to suffer the outbreak. However, it is notorious that the time interval between the first patient and containment is not being proportional between countries. This derives directly from the interaction of four factors: the confinement measures applied by the country, the epidemiological period in which they are applied, the country's own idiosyncrasy, and the population susceptibility to SARS-CoV-2 haplotype/s with presence in their country.
The worldwide SARS-CoV-2 epidemic has brought about a change of social paradigm. The fact that it is not a lethal disease has allowed updating the protocols in the face of a new pandemic. However, in light of the abundant latest scientific research results, much information remains to be integrated. Some of the open investigations are; in principle, futile, since they seek to obtain scientific revenue at the cost of sacrificing quality of research. This can be a problem, but also a virtue. It allows a huge variability of sources to exist, which enable us to study specific areas of this disease in a much richer and more prolific way.
In this review we have described some of the most characteristic facts of this pandemic, to relate them to the physical-chemical structure of SARS-CoV-2, a topic that has not been explored too deeply. In light of current events, we conclude that there is sufficient evidence to believe that SARS-CoV-2 possesses unique ultrastructural characteristics, derived solely from its unique evolutionary pattern and efficient coevolution with human populations and environmental adaptation. Studying the changes of these characteristics should yield knowledge on the evolutionary pattern of SARS-CoV-2 related viruses in the near future. The vaccine should appear early to contain SARS-CoV-2 diversification before existing haplotypes generate three remarkably different lineages. More research is required to analyze the possible uses of SARS-CoV-2 in directed evolution studies.
Received on 29.05.2020 Modified on 21.06.2020
Accepted on 19.07.2020 © RJPT All right reserved