Matteo Chiara^1,2, Barbara Illi³, Graziano Pesole^2,4
1Department of Biosciences, University of Milan
²Institute of Biomembrane Bioenergetics and Molecular Biotechnologies, National Research Council (IBIOM-CNR)
³Institute of Molecular Biology and Pathology, National Research Council (IBPM-CNR), c/o Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome.
⁴Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “A. Moro”

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Table of Contents

Last update: 2020/20/09

• Introduction
• Emergence of a zoonotic pandemic
• Coronaviruses and zoonoses
• 2002: SARS
• 2012: MERS
• From bats to humans
• SARS-CoV-2: bat or pangolin?
• SARS-CoV-2: artifical or natural?
• Mutation capability and SARS-CoV-2 variants
• Potential dangerous mutations: the RdRp variant
• Potential dangerous mutations: the D614G variant
• Conclusions

For “non-experts”

• Allele and allelic frequency

References

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Introduction

Three conditions have to be satisfied for a zoonosis to spread and become pandemic. First, the virus has to be compatible with humans. Second, a close contact among animals and humans has to exist. Third, once a leap of species has occurred, the virus has to be transmissible among humans. Unfortunately, COVID-19 pandemic satisfies all these three conditions. However, it is still not completely clarified from which animal the SARS-CoV-2 virus (hereafter simply CoV-2), the etiological agent of COVID-19, has derived.

A high number of studies have attempted to identify the prerequisites required and the environmental, geographical and ecological conditions which may favour pathogens spillover events¹. Determining these factors is the key to foresee and prevent possible pandemics and outbreaks in the future. Despite numeorous efforts, our knowledge of the ecology of possible new pathogens is almost incomplete. In a recent paper, Woolhouse and collegues have classified and descrived about 1339 different human pathogens, among them 87 – mostly viral – emerged only since 1980². The authors report for each decade an increasing number of potential spillover events. Therse are generally localized in regions of our planet with specific environmental, ecological, social, economical conditions, which are defined hotspots.

Emergence of a zoonotic pandemic

A zoonotic pandemic emerges when a pathogen which could previously infect only animals begins to infect humans. Three steps may be identified:

1. During the first step (pre-emergency), upon environmental and demographic changes, the pathogen meets other species and expand its trajectory.
2. During the second step (localized emergency) the contact with animals or products of animal origin promotes the possible transmission to humans. As humans are new hosts, the pathogen cannot efficiently transmit among individuals.
3. During the third step (the onset of a pandemic), the pathogen transforms and specializes. Now, it is able to support long chains of transmission also among humans. A sequential series of transmission events takes place. Global journeys and other human activities facilitate the transmission in new geographic areas.

Hotspots are regions with elevated wildlife bio-diversity and which have been recently subjected to demographic changes due to human activities, as an uncontrolled increase of agricultural and zootechnical activities1. Furthermore, a global analysis of emergent human infectious diseases suggests that these pathologies find a fertile ground for their diffusion because of a failure of public health and surveillance systems. Based on these considerations, it is evident that human actions to prevent and monitor emergent infectious diseases should be mainly focused on areas of our planet with elevated bio-diversity in which phenomena of demographic expansion are not supported by public health infrastructures and by dedicated funds to zoonoses control³.

According to recent studies, east and sud-east Asia, India and equatorial Africa, are the most dangerous regions for the development of new zoonoses^1,4. In the past few years, the monitoring activity by metagenomic sequencing has identified an increasing number of new viruses or pathogenic agents with possible zoonotic potential, but predicting which of these new entitites may represent a risk for the public health now and in the future is not easyIt is well established that many viruses with zoonotic potential circulate in their natural reservoirs for a long time4. Their spread from natural hosts ti humans and other animals is alrgely due to human activities, including the actual agricultural practice and urbanisation. Therefore, the most efficient way to prevent viral zoonoses is to maintain barriers between natural reservoirs and human society, taking into account the concept of “global health”.

Coronaviruses and zoonoses

«Everything that happens once may never happen again. But everything that happens twice will certainly happen a third time»
Paulo Coelho, The Alchemist

Coronavirus are single-stranded RNA viruses. According to the International Committee for Virus Taxonomy (ICTV), they belong to the Coronaviridae family of Nidovirales order. The coronavirus sub-family is composed by 4 genus: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Delatacoronavirus. Alpha and Betacoronaviruses infect only mammals. Gamma and Deltacoronaviruses infect birds but some of them may infect also mammals⁵. Alpha and Betacornoaviruses usually cause respiratory syndromes in humans and gastroenteritis in animals. Besides highly pathogenic coronaviruses, SARS-CoV, CoV-2 e MERS-CoV, which cause a severe respiratory syndrome in humans, other four coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43 e HKU1) may infect humans, but in the vast amjority of cases, induce only mild diseases of the upper respiratory airways. Alphacoronaviruses and Betacoronaviruses may elicit acute diseases in some animal species of zootechnical interest, such as the swine transmissible gastroenteritis virus⁶, the porcine epidemic diarrhea virus (PEDV)⁷ and the swine acute diarrhea syndrome coronavirus (SADS-CoV)⁸.

All the coronaviruses that are able to infect humans are emerged from zoonotic events. SARS-CoV, CoV-2 MERS-CoV, HCoV-NL63 e HCoV-229E probably stemmed from bats whereas HCoV-OC43 e HKU1 stemmed from rodents⁹. Domestic animals may have important roles as intermediate hosts which allow virius transmission from natural original hosts to humans. Furthermore, the same may suffer from diseases caused by coronaviruses transmitted from bats. This is the case of PEDV and of SADS-CoV: both represent a recent spillover from bats to swines⁹. As 7 of 11 Alphacoronaviruses species and 4 of 9 of Betacoronaviruses species have been identified only in bats to date, it is believed that bats are the main natural reservoir of Alpha- and Betacoronaviruses⁵.

Before CoV-2 outbreak, the 21st century already knew the global spread of other two coronaviruses, previously unknown.

2002: SARS

On november 2002, in Foshan, China, the first case of a severe acute respiratory syndrome (SARS) was reported¹⁰. During the following months, in continental China new cases arose and on february 2003 SARS cases scaled up to 300. Thereafer, travels have spread the outbrak to Hong Kong and from Hong Kong to Vietnam, Canada and many other countries¹¹. On march 2003, the WHO constituted a laboratory network to isolate SARS etiologic agent. A remarkable global effort led to the identification of SARS coronavirus (SARS-CoV) at the beginning of april 2003. The outbreak ended on july 2003, after arriving in 27 countries with a total of 8.096 confirmed cases and 774 deaths. Five more SARS cases, derived from a strictly localized zoonotic event, have been detected on december 2003-january 2004¹³. From then, no other SARS cases have been detected. Prevention and control measures against infections – such as quarantine and social distancing – have been fundamental to the SARS outbreak. However, in the following years, several studies have highlighted how SARS-CoV-related viruses, potentially capable to infect human cells, kept to circulate among bats¹².

SARS-CoV isolation in palm civets, badgers and raccoons in a market of living animals in Shenzen, China¹³, provided the first indication about the possible origin of this new virus. However, subsequent analyses have demostrated how these animals provided only incidental hosts, because the presence of viruses similiar to SARS-CoV has never been reported in palm civets or in farm animals¹⁴. Only in 2005, thanks to a punctual research work and environmental sampling, SARS-CoV-related viruses have been isolated in bats from Rhinolophus genus. This finding was pivotal to identify the natural reservoir of the new coronavirus. Thereafter, many SARS-CoV-related coronaviruses (SARSr-CoV) have been found in bats in many areas of China and also in European, African and South-Est Asia regions¹⁴. These data indicate that SARSr-CoVs have a large geographic spread and that could have been present in bats since a long time.

2012: MERS

The onset in 2012 of the middle est respiratory syndrome (MERS) has introduced the second, highly pathogenic, coronavirus into the human population in the 21st century. On june 2012, ten years after the SARS outbreak, a man in Saudi Arabia dead by acute pneumonia and renal failure. A new coronavirus, the middle est respiratory syndrome coronavirus (MERS-CoV), was isolated from his sputum¹⁵. Afterwards, a series of abnormal cases of severe respiratory disease, occurred on april 2012 in a hospital in Giordania, has been retrospectively diagnosed as MERS¹⁶. MERS-CoV continued to emerge and spread in countries aouside the arabic peninsula because of travels of infected individuals. Frequently, these imported MERS cases provoked the spread of the virus in hospital environment. For example, on may 2015, a single individual back from Middle East has started a hospital outbreak in South Corea which has involved 16 hospitals and 186 patients. To date,the known MERS cases worldwide have been little less than 1800 in 27 countries, with 624 deaths. Differently from SARS-CoV, MERS-CoV is believed to have few possibilities to be directly transmit among humans. The majority of the known MERS cases is the result of independent zoonotic spillover events from camels or dromedary to humans. The scarce transmissibility of MERS-CoV among humans, coupled with disease high death rate, is one of the factors that has limited the spread of MERS.

Based on the previous knowledge on SARS-CoV, the reasearch of MERS-CoV natural reservoir focused on bats first. Serological testing on samples from dromedary and camels in Oman and Canary Islands reveald a high prevalence of anti-MERS-CoV antibodies in these animals¹⁷, suggesting an alternative scenario. Retrospective analyses of oro-pharingeal swabs from dromedaries and camels in a farm in Qatar, linked to 2 cases of human MERS, allowed the isolation of the virus from dromedaries and camels in Saudi Arabia and Qatar¹⁸. On the basis of theses data, the most plausible model for MERS virus transmission from animals to humans is that, at the beginning, the virus spread from bats – the natural reservoir – to dromedaries and camels which provided the intermediate hosts. Given the zootechnical importance of these animals in many areas worldwide, the continuous proximity of infected camels and dromedaries may explain the numerous MERS-CoV zoonotic transmission events to humans.

From bats to humans

Bats are the natural reservoir for different coronaviruses, including SARS-CoV and MERS-CoV. SARS-CoV has crossed the barrier among species spreading from bats to palm civets and/or other living animals which are usually sold in China wet market. The genomic analysis suggests that phenomenon occurred at the end of 2020. Many people in contact with palm civets have been exposed to SARS-CoV. An ancestor of MERS-CoV spread from bats to camels and dromedaries; serological analyses indicate that this happened more than 30 years ago. The continuous circulation of MERS-CoV in camels and dromedaries cause a frequent zoonotic transmission of this virus. SARS-CoV and MERS-CoV spread among humans mainly through contagion chains limited to hospitals.

SARS-CoV-2: bat or pangolin?

The first COVID-19 cases have been initially linked to the wet market Huanan, in Whuan, in which, besides fruit, vegetables, fish and fresh meat, also living animals are sold. Since CoV-2 has undoubtely a zoonotic origin, a relationship with this activity should not surprise. However,because of not all the first cases are linked to the Huanan market, it is possible that the sequence of events that led to the spread of CoV-2 to humans is more complicated than initially suspected. The analysis of all the environmental samples obtained from the market reveals that all the isolated viruses are strictly related to the first Wuhan patients. Even if this data suggests again that the market played an important role in virus spread, it is not clear whether the samples belonged to infected people, who accidentally deposited infected material, or from animals or from animal material present in that place. Unfortunately, since Chinese authorities have not performed any kind of direct sampling from animal species in the market, it is difficult, maybe impossible, to make any other further consideration.

The first information on CoV-2 origin came from the analysis of the bronchoalveolar lavage fluid (BALF) of one of the seven patients affected by severe pneumonia admitted in the intesive care unit of Jin Yin-Tan hospital in Wuhan¹⁹. Databank search to identify similarities among that isolated viral genome and known viral genomes indicated that the virus eas very similar to SARSr-CoVs. Further analyses demonstrated that the virus probably derives froma bat coronavirus, in particular from the TG13 coronavirus which infect the Rinolophus affinis bat (Bat-CoV-RaTG13).

That CoV-2 strictly related viruses came from bats was not a surprise. Bats are the reservoir species for a wide group of coronaviruses¹⁴. The detailed work of monitoring performed in China in the past few years has identified a high number of bat coronaviruses, including RaTG13. However, the precise role played from bats in the zoonotic origin of CoV-2 is not clear. Curiously, CoV-2 most similar viruses have been sampled in the Yunnan province, 1.500 km far from Wuhan¹⁹. This probably means that, despite the great efforts, our knowledge of bat coronaviruses is still largely incomplete. Moreover, even if sequence similarities between RATG13 and CoV-2 (in a range from 95 to 96%) indicate that these viruses are strictly linked, they show some differences that probably correspond to an evolution occured in more than 20 years20. Therefore, a more accurate sampling will identify other bat viruses which are even more strictly related to CoV-2. Hence, several years may be required to precisely identify the chain of events that allowed CoV-2 spread to humans.

As previously underlined, bats are the larger natural reservoir for coronaviruses. However, these animals occupy an ecological niche well separated from humans. Therefore, it is highly possible that other mammals privede the intermediate host, in which CoV-2 was able to acquire the mutations required to efficiently spread among humans. To identify these putative intermediate hosts, it is imperative to rigorously study which kind of coronovairuses may circulate in animals in strict contact with humans.

Indeed, strictly CoV-2-related viruses have been found in pangolins, which are illegally imported from Guandong prvince and sold in chinese markets. the metagenomic analysis of lungs from two pangolins, dead in a time frame compatible with COVID-19 outbreak, revealed that those pangolins were infected by a virus (after defined Pangolin-CoV) strictly related to CoV-2 and other bat SARSr-CoV21. CoV-2 and Bat-CoV-RaTG13 genomes are basically identical to the genome of PangolinCoV (see figure 2B in Zhang et al., Current Biol, 2020).

Pangolin-CoV and CoV-2 share in the spike protein sequence 5 key amonoacids to enter host cells. This makes CoV-2 spike gene more similar to the corresponding (homolog) gene in Pangolin-CoV than in Bat-CoV-RaTG13, suggesting that mutations may occured in the pangolin vrus and that pangolins represented the intermediate hosts. However, the comparison of the genomic sequences seems to indicate PangolinCoV as the common ancestor of both CoV-2 and Bat-CoV-RaTG13 and together these 2 viruses could constitute a new group fo coronaviruses, termed SARS-CoV-2 group. However, the pangolin origin of CoV-2 is still debated. In fact, because coronaviruses are characterized by (relatively) low mutation rates, with respect to other RNA viruses, and carry in their genomes evident signs of recombination, a process which mix the genetic material, the phylogenetic analyses to determine the precise relationships among coronoaviruses – including CoV-2 – are highly complex and frequently not definitive9. Moreover, while our past experience with coronaviruses suggests that the evolution in intermediate hosts is required to infect humans, it cannot be excluded that the virus acquired some of its key properties during a period of “cryptic fusion” in humans.. Indeed, the virus could have circulated in human populations well before december 2019 (and maybe not in Wuhan) and that it was not detected becausi of asympotmatic infections or with mild respiratory symptoms. Also sporadic pneumonia cases were not recognisable using the standard methods normally applied for the surveillance and identification of pathogens. During this period of cryptic transmission, the virus could acquired step by step key mutations which allowed its complete adaptation to humans.

SARS-CoV-2: artifical or natural?

The idea that CoV-2 could be a lab-created, artificial virus has greatly destabilized public opinion. A video circulating on the social media, related to an italian press report about a synthetic SARS coronavirus (SARS-CoV), has immediately raised doubts about CoV-2 origin and about China responsibility in its spread. Nevertheless, that video, which was within a program called Leonardo, dates back to 2015 and, specifically, reports the construction, by a method called cloning, and characterization of a chimeric virus in which the spike SHC014 protein of a bat coronavirus was inserted into the backbone of a mouse SARS-CoV22. In particular, the SHC014 gene was cut from an available plasmid and pasted into a mouse SARS-CoV, previously in the same way, to permit a correct SHC014 insertion.

In that paper, in vitro and in vivo studies demonstrated the inefficacy of both anti-SARS-CoV neutralizing antibodies and vaccines, respectively, to slow virus replication within host cells and to protect mice from infection. Therefore, a great alarm raised when Leonardo press report was re-published in last March. However, that work was simply a proof of principle that a SARS-CoV-derived virus and related pandemic could have raised and been dangerous for the human being due to the potential lack of therapies and vaccines. Indeed, SARS-CoV-2 has no traces, in its genome, of any mouse SARS-CoV sequence.

Another concern raised about the presence of four small aminoacidic sequences in Cov-2 spike protein, the tool the virus uses to enter human host cells, common to the Human Immunodeficiency Virus (HIV). Such similarity was recently stressed by Luc Montagnier, one of the “fathers” of HIV, strengthening the hypothesis of Cov-2 as the product of a genetic manipulation. However, those sequences are common to about other 300 unrelated proteins.Further, on february 2020, the comparison of CoV-2 genome with other other available genomes clearly indicated CoV-2 did not acquired those sequences from HIV23. More recently, a Correspondence on Nature Medicine Journal, has well clarified that CoV-2 was the product of a natural selection of zoonotic viruses²⁴.

Mutation capability and SARS-CoV-2 variants

With the development of COVID-19 outbreak more viral genomes have been sequenced and this allowed the study of CoV-2 evolutionary process. Given the recent common ancestry, the first Wuhan samples contained poor geentic diversity. In more recent time windows, in different areas worldwide, possible virus variants have been identified. Despite the growth of genetic diversity means that now we are able to detect different CoV-2 types, it’s still difficult to determine, with genomic comparisons only, whether the virus is accumulating mutations relevant from an epidemiological point of view, while spreading in the human population around the world and any kind of assessment requires a careful scientific validation.

Survival and fitness maintenance underlie the high mutation rate of RNA viruses, such as Coronaviruses. When bottleneck events occur and viruses slow their replication rate they lose their fitness and deleterious mutations for their survival may appear. Conversely, repeated and large population passages result in rapid fitness gain.
Given the high mutation rate of RNA viruses, it is obvious that many mutations in the viral genomes will appear and these mutations will help us to trace CoV-2 spread. However, it is increasingly clear that, because of the current dimensions of the pandemic, the sample of genomic sequences available will be always limited with respect to the total number of cases and that it will be difficult or even impossible to reveal the single chains of transmission. Despite coronaviruses have probably low mutation rates in the short period with respect to other RNA viruses, in the long period their nucleotides substitution rate (that is the speed with which their genome evolves) is too different from the nucleotides substitution rates observed in other RNA²⁵. This suggests that low mutation rates are somehow compensated by high replication rates inside the host cells. In fact, a large viral population may easily compensate the reduced rates of molecular evolution. There are no proofs that this mutation ability (common among RNA viruses) has a radical impact in virulence and transmissibility, in particular in small time frame. However, it is important to monitor possible changes on CoV-2 phentoype, as long as the virus spreads. Currently, it seems logical to foresee that probably any decrease in COVID-19 cases will be due to the increase in the immunity of the human population and to the epidemiological context, rather than to an evolution of the virus.

RNA viruses can tolerate a low number and few types of mutations²⁶. Furthermore, it has to be considered that a large part of mutations are “synonymous” or “silent”, that is the change in the RNA sequence does not correspond to a change in the corresponding aminoacids encoded by that specific RNA sequence. A study on 95 CoV-2 genomes revealed that all of them were similar at 99.9% both at the DNA and protein levels.

Nevertheless, 13 selective genomic sites have been discovered in CoV-2 with high mutation rates. Importantly, some “hot-spot” mutations occur in the genes encoding for spike and for nucleocapsid proteins and this is particularly relevant for viral replication, transmission and induced immunity related studies²⁷.

Mutation studies have been important for the detection of CoV-2 variants, which have been found to characterize specific geographic areas. For example, the analysis of 160 CoV-2 genomes worldwide has permitted to identify three principle types of CoV-2, marked by specific aminoacid changes, termed A, B and C²⁸ and characterized by particular aminoacid variants.

• Type A, which is further subdivided in two subgroups by the synonymous mutation T29095C (T=thymine; C=cytosine; 29095 is the nucleotide position in the genome), spread mainly in China and less in East Asia, Europe, USA and Australia.
• Type B is derived from A, because of two mutations, one synonimous (T8782C) and one non-synonimous (C28144T), this latter changing the aminoacid leucine to serine. It is interesting to note that type B has left East Asia after the accumulation of other mutations.
• Type C differs from type B by a non-synonymous mutation (G26144T; G=guanine) which changes a glycine into a valine.

Interestingly, these analyses showed that, apparently, here in Italy we had one entry of type B-derived CoV-2 from Munich and one earlier, type C-derived, from Singapore. This kind of analyses are particularly important to reconstruct infection paths. However, this is a picture of the pandemic at early stages.

Recent studies, based on an higher number of viral genomes, show that it is possible to identify an increasing number of strains which differ at the genomic level^29,30. Applying the phylogeoraphic discipline, which consists in the study, overtime, of viral evolution in different areas worldwide, different resarchers identified some of the fundamental characteristics in the evolutionary process of CoV-2³¹. For example, many studies, comparing CoV-2 genomes with a regular frequence, in different time frames, measured the evolutionary speed of the virus, a fundamental parameter to understand at best CoV-2 adaptation potential to the human host. The comparison of the genetic distance among CoV-2 genomes showed a number of differences very close to the number of differences typical of other coronaviruses and almost identical to the values observed for SARS-CoV^20,29,32. On the basis of the mutations observed and on the evaluation of the viral evolutionary speed (1,84 · 10^-3 substitution/site/year), researchers have evaluated the time window in which the first transmission to humans of the new virus occurred. Regarding this issue, many studies agree in confirming that CoV-2 circulated among humans at least since last september, therefore, well before the viral pneumonias outbreak in Wuhan.
Considering the whole pool of genomes available, we know about 8000 different mutations in CoV-2 genome. 99,99% of these mutations has an allelic frequence lower than 1%, that is it is observed in less than 1% of the known sequences. Only 50 mutation show a frequence identical or higher to 1%.

As previusly discussed, detailed comparisons of all the avilable genomes (82000 on august 15^th, 2020) indicate that, currently, using the genomic variability data, it is possible to distinguish different CoV-2 genomes³⁰. It is very interesting to note that all the genomes identified to date show a very different prevalence in several areas worldwide. This observation has been object of a long debate during the outbreak phases. It has been hypothesized in several occasions that the different fatal rates found in different areas could be associated to the higher or lower prevalence of viral strains. These observations have not been validated yet, and the estimate of fatale rate worldwide are, more likely, due to the application of different prevention/containment policies and/or of different diagnostic methods in different areas of our planet³³.

It is important to note, however, that the prevalent viral type in Europe, South America and some regions in North America shows characteristic mutations which are not present in prevalent viruses in China. Two of these mutations have been object of thorough studies, to verify the possible functional effects.

Potential dangerous mutations: the RdRp variant

One of the mutations which charachterizes viral isolates outside China deserves a particular attention, because it is in the RNA-dependent RNA polymerase (RdRp) gene³⁴.

RdRps from di SARS-CoV and CoV-2 are very similar and this indicate that coronaviruses try to maintain this protein conserved in its structure and function. RdRp mutation, at the nucleotide 14 408, appeared in England on Febrary 9^th 2020. In parallel, a dramatic increase in COVID-19 cases occurred in Europe. More importantly, starting from that day, according to a prelimiary analysis based on a relatively low number (220) of genomes, a possible increase in the mutation frequency of other viral proteins encoded by the genomes with mutated RdRp with respect to RdRp non-mutated genomes, was observed. This mutation should not compromise the replication catalytic activity, but seems to impair the binding with other non structural proteins, such as nsp14, which is homolog to nsp14 form SARS-CoV and that should have an editing activity, that is corrects the errors that RdRp may introduce during the replication process, removing the wrong nucleotides and inserting the correct ones.

This hypothesis may explain at the molecular level the increase in the mutation frequency in other genes (and proteins), as a consequence of replication errors. Furthermore, this site is very close to the RdRp interaction pocket with antiviral drugs which block its activity, such as filibuvir and tegobuvir. Therefore, it will be important to validate whther this mutation effectively compromise the fidelity of the replication process and may arise drug resistance phenomena. it is important to underlie, however, that in the lack of experimental evidences, these theories have to be considered hypotheses, and have to evaluated with care. In fact, according to more recent studies, based on the sampling of an higher number of genomes (more than 20.000), the mutation rates of CoV-2 type currently circulating are not significantly different. These results suggest, as reported by other sources^20,29,30, that at the moment there are no solid evidences about viral variants with faster mutation rate. As highlighted before, only the availability of an ever higher number of genomes and the possibility to perform lab experiments in the future in controlled conditions, will allow to verify whether and how the RdRp mutation identified by Pachetti and collegues is capable to make CoV-2 evolution faster.

Potential dangerous mutations: the D614G variant

A second mutation in CoV-2 genome attracted research attention worldwide. As RdRp mutation, it is present mainly in CoV-2 genomes isolated in Europe (but also in South America and some regions in North America). The A23403G mutation correspond to an amino acid substitution in the spike (S) protein of the aspartic acid (D) 614 with a glycine (G) (D614G)³¹.

S protein, which extend from virion surface, confering the typical crown shape to coronaviruses, mediates virus fusion with the membrane of the host cells, a fundamental event which allows the virus to enter and infect the cells. Because of its relevance for the development of therapeutic routes and vaccines, monitoring S gene nutations iso ne of the main of the scientific community.

During these monitoring activities, several researchers have found that the D614G variant of S protein became the most prevalent circulating virus phenotype. The dynamic assessment of the allelic frequencies revealed that the increase of G614 frequency happened at many geographic levels: national, regiuonal and municipal and regularly³¹. This change occurred also in local outbreaks in which the D614 phenotype was prevalent before G614 variant introduction, suggesting that this latter may confer an advantage to the virus in terms of fitness.

Molecualr modeling analyses indicate that the 614 residue is located on S superface and the change from D to G may result in a more flexibility of the protein, which could enhance virus infection capability. Moreover, this substitution could modulate the glycosylation of the near N616 site, a bio-molecular reaction requie for the recognition of the virus by our immune system.

Two independent studies revealed that in several cellular models, the virions with the G614 variant may reach concentrations from 2 to 9,3 higher than the virions with the D614 phenotype31. Furthermore, the G614 variants is associated to elevated COVID-19 patients viral loads, suggesting a higher virus infectivity. Despite these observations, no correlation has been found between the G614 S variant and the severity of the disease to date. The epidemiological relevance of G614 S varianti s not completely clear. However, it is evident that the emerging monitoring strategies of new mutations are pivotal to udnerstand the olecular mechanisms which rule COVID-19 pathogenesis. Sequencig-based approaches and the comparison of an increasing number of genomes will be a precious tool in the future to acquire a more comprehensive knowledge of this new virus.

Conclusions

Since its appearance, CoV-2 mutated many times and now we are able to distinguish different virus variants^28,30. However, despite during the different phases of the outbreak some reaesearchers proposed that sone of the new viral phenotypes, prevalent in different areas worldwide, may result in a more aggresive pathology, no relationships have been descrive between the viral and the clinical phenotype. The fatality rate in specific geographic areas also has not been related with the rpesence of particolar virus variants, while the differences in the fatality rate seem to correlate in a significative manner with different demographic and socio-economic markers³³.

It has been already demonstrated that RNA viruses may accumulate mutations overtime which may confer a higher pathogenicity6. From this point of view it is important to underly that some CoV-2 mutations, which ate prevalent in Europe may explain, at least in part, the higher incidence of COVID-19 in pur continent^31,34, even f at the moment we still do not have valid and definitive proofs. Therefore, in the future it will be important to monitor the occurrence of new mutations in CoV-2 genome and to perform focused lab experiments to understand in a comprehensive manner the possible functional implications of the emerging mutations. Nevertheless, thorough considerations about the CoV-2 zoonotic origin demonstrate that dynamics through which the new virus adapted to humans are probably similar to those of SARS e MERS outbreaks. In this regard, we may take the risk to say that the current pandemic does not represent an unpredictable event. From these premises and the constant anthropic activity of colonization of inaccesible and barely popolate areas, it is highly probable that the contamination of urban areas with the wild ones will allow spillover events of viruses rom other animals to humans with an ever more high frequency. For this reason, prevention and surveillance strategies of possible zoonotic events have to be implemented also in the basis of the acquired knowledge during this pandemic.