Article title: Hypothesis of Potential Evolution of SARS-CoV-2 through Hybridization of SARS-CoV-1 with Saccharomyces cerevisiae and Mycobacterium avium naturally inside an immunocompromised Pangolin


 
 
 
 Abstract 
 
 
 
 
 Background
 
 Discovery of the origin of SARS-CoV-2 become an urgent international need due to the current health and economic implications. Recently, pangolins were reported to have a diminished immunity and vulnerable to several infections. By the end of 2019; recent studies reported the pangolins infection with SARS like virus that showed a 99% genetic homology with SARS-CoV-2. In 2020; a genetic mutation was reported in pangolins that increases their vulnerability to infection with flagellated bacteria such as Mycobacterium species. Moreover, the introduced food formula containing yeast at several zoos, considered as a source of opportunistic 
 Saccharomyces infection in such immunocompromised pangolins.
 
 
 Methods
 
 The coexistence of multiple infections in an immunocompromised pangolin is the concept of SARS-CoV-2 evolution hypothesis. The structures of the three proposed microorganisms; SARS-CoV-1, 
 Saccharomyces cerevisiae and
 Mycobacterium avium as well as their glycobiology and host cell interactions were studied. The
 α-mannoses and phosphates present in the spike protein of SARS-CoV-2 is supposed to be introduced through fermentation of the spike protein of SARS-CoV-1 by 
 Saccharomyces cerevisiae while the PRRAR furin putative sequence as well as the LDPLSE
 motif are supposed to be introduced
 through microbial interaction with 
 Mycobacterium avium strain 104
 . Moreover,
 the interaction with pangolins cells resulted in the presence of the reserved O-glycosylation sites in the spike protein of SARS-CoV-2.
 
 
 Results and discussion
 
 SARS-CoV-2 was naturally developed in an immunocompromised pangolin through microbial interaction that resulted in a hybrid microorganism containing viral RNA encapsulated within a glycan shield (Spike glycoprotein) manipulated by both 
 Saccharomyces cerevisiae and 
 Mycobacterium avium.
 
 The proposed origin of the novel virus; SARS-CoV-2, explained the abnormal clinical findings such as disseminated infection, blood clotting, hyperpigmentation in some cases and Kawasaki like syndrome in kids. Lactose intolerance is also suggested as a new genetic risk factor that increases the vulnerability to infection with SARS-CoV-2. Moreover, the likelihood biofilm formation by SARS-CoV-2 is discussed. Furthermore, this hypothesis discussed the potential evolution of MERS through microbial interaction between 
 Mycobacterium tuberculosis and SARS-CoV-1 virus.
 
 
 Conclusions
 
 Trials using triple therapy treatment strategy consisting of Remdesivir, Aithromycin and fluorocytosine as well as a yeast-based vaccine are suggested.


Background
In December 2019, a cluster of pneumonia cases epidemiologically linked to the Huanan seafood market in Wuhan city (Hubei Province), China [1,2] were reported. In early January, the etiological agent of the pneumonia cases was found to be a coronavirus that was subsequently named SARS-CoV-2 [3][4][5].
The first genomic sequence of SARS-CoV-2 was reported on January 10, 2020 [6]. A phylogenetic comparison of SARS-CoV-2 with other CoVs showed that bats were considered as the native host of SARS-CoV-2 as the new virus is 96% identical to two SARS-like CoVs from bats called bat-SL-CoVZX45 and bat-SLCoVZX21 [7]. Researchers in Guangzhou, China, suggested that pangolins are the potential intermediate host of SARS-CoV-2 based on 99% genetic homology in a CoV discovered in pangolins and SARS-CoV-2 [8].
Figuring out the original source of SARS-CoV-2 become an international demand because of the current health and economic implications. In outbreaks of zoonotic pathogens, identification of the infection source is crucial, as this may allow health authorities to separate human populations from the animal reservoirs posing the zoonotic risk [9,10]. Identification of the exact source of the virus is important to answer the outstanding evolutionary question about whether the virus was laboratory manipulated or naturally developed. Moreover, it is of a great benefit while developing a vaccine and/or treatment.
A well-known model for microbe-microbe interaction is the human microbiota where commensal microorganisms (bacteria, archaea, viruses, and microscopic eukaryotes) are engaged in a constant crosstalk together and with the host immune system and metabolism [11].
One of the recent studies discussed the evidence of recombination in coronaviruses implicating a pangolin origin of nCoV-2019 [12]. Another recent mathematical study found an evidence of recombination event at least 11 years ago in an ancestor of the SARS-CoV-2 involving the RBD and stated that SARS-CoV-1 and SARS-CoV-2 share a similar genotype in RBD [13] that could show the fact of SARS-CoV-2 is a recombination product of SARS-CoV-1. This proposed fact come in accordance with the hypothesis to be discussed in that article.
The yeast Saccharomyces cerevisiae has been used as cell factory for production of several biopharmaceuticals proteins such as insulin, human serum albumin, hepatitis vaccines and virus like particles used for vaccination against human papillomavirus [14]. The ability of yeast to change the structure of SARS virus has been previously described in a research that stated the high-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris [15]. More recent research illustrated the ability for re-construction and cloning of the viral genome RNA of SARS-CoV-2 in one step using Saccharomyces cerevisiae [16].
Pangolins are unique placental mammals which have adapted to a highly specialized diet of ants and termites, and are of significance in the control of forest termite disaster [17]. Besides their ecological worth, pangolins are very necessary economic animals with the worth as drugs and food. Pangolin meat is cooked in lees (dead yeast and debris) and sold in China as a street food during the winter [18]. Additionally; the Chinese traditional medicine prescribes the pangolin scales for a variety of ailments such as excessive anxiety in children, women thought to be possessed by devils, malarial fever, and deafness [19]. Pangolins are traditionally used in Chinese medicine for promoting menstruation and lactation, reducing swelling and promoting discharge of pus in abscesses and boils and for rheumatism/arthritis [20]. Moreover, there are a lot of Chinese patents registered for treatment of different types of tuberculosis based on the pangolin scales as a medicine for treating lymphatic tuberculosis [21,22], mammary glands tuberculosis [23-25], tuberculosis in bone and joint [26,27], lung tuberculosis [28,29].
Recently, captive breeding is an important way to protect pangolins from extinction due to illegal hunting and habitat destruction [17], but there are several technical barriers to successful captive breeding programs such as pangolin's high dependence on natural ecosystem and availability of suitable accommodation and artificial diet. The Taipei Zoo developed a food formula to improve the food intake and digestive disorders that contains a high amount (2 g) of yeast powder [30]. Because of pangolin's poor adaptability to captive environment and a weak immune system, over 50 percent of captive pangolins died of hemorrhagic gastric ulcers and pneumonia [31]. Skin diseases and parasitic infection (ticks and mites) are also common in pangolins especially for those seized from illegal trade due to the mess and dirty transportation process [17,32,33]. These parasites probably cause pangolins to be anemic, wasting, immunocompromised and vulnerable to infectious disease, and even death [17]. Another recent study in 2020 stated that a genetic mutation happened in pangolins results in convergent losses of TLR5 (Toll like receptors) that play an important role for the innate immune system. TLR5 encodes the major extracellular receptor for detection bacterial flagellin [38] and as a result this could increase the risk of pangolin infection with a flagellated bacteria such as Mycobacterium species.
In this hypothetical study, it is proposed that SARS-CoV-2 originated in an immunocompromised pangolin co-infected by SARS-CoV-1 like virus, Saccharomyces cerevisiae and Mycobacterium avium strain 104 through microbe-microbe interaction that led to generation of the novel virus; which is a virus containing both fungal and bacterial epitopes.

Methods
(i) Study of the spike glycoprotein of SARS-CoV-2 compared to SARS-CoV-1, (ii) Hypothesis of hybridization of SARS-CoV-1 with Saccharomyces cerevisiae and Mycobacterium avium, (iii) Comparative study for the diseases pattern caused by SARS-CoV-2, Saccharomyces cerevisiae and Mycobacterium avium (i)

Study of the spike glycoprotein of SARS-CoV-2:
Since COVID-19 outbreak in December 2019, scientists all over the world started to study the structure of the new virus comparing it to the previous human coronaviruses such as SARS, MERS and HKU1 CoVs [39][40][41]. Most studies tried to Figures out the structure of spike protein which is reported to bind to ACE 2 receptor [42,43] as well as CD209L, a C-type lectin [41].
There are several unique features discovered in the structure of the spike (S) protein of SARS-CoV-
Another important issue to be noted about oligomannose-type glycans in SARS-CoV-2; is their ability to bind to C-type lectin (CD209L) through α-mannose residue to mediate the endocytosis of pathogens indicating the alpha configuration of mannose sugar in glycoprotein of SARS CoV-2 in contrast with the beta mannose in SARS-CoV-1 [41].
(5) Phoshphorylation: SARS-CoV-2 S1 protein was examined and 36 positions of Ser/Thr residues were found to be phosphorylated on almost all domains of the protein. Some phosphorylation positions were neighbors to the conserved O-and N-glycosylation sites on the 3D structure [41] .
(6) LxxIxE-like motif: a recent research detected the presence of LxxIxE-like motif (LDPLSE ) in S1subunit that could recruit the host protein phosphatase 2A (PP2A) [46] and this motif is conserved in SARS-CoV-2 and LIDLQE in S2 subunit which is conserved in S2 subunit of SARS-CoV-2, SARS-CoV, SARS-like of bat from China and Kenya [46].
"The base of this hypothesis depends on the assumption that the spike protein of the SARS-CoV-1 (assigned as CoV-A) virus is highly mannosylated (Man5GlcNAc2 to Man9GlcNAc2) glycoproteins. In a glucose deficient medium such as inside pangolin's macrophage cells, the mannose sugar in CoV-A glycans could be utilized by Saccharomyces cerevisiae for its energy production as well as mannosylation reactions. The microbial reaction started with endocytosis of CoV-A inside yeast cells followed by phosphorylation of the spike glycoprotein of CoV-A (to stop endocytosis) and then fermentation of CoV-A mannosyl glycan. At this point, the virus is naked from most of its glycan shield (assigned as CoV-B) and in a trial to rebuild its shield, it enters the Golgi apparatus of S. cerevisiae and makes use from the yeast specific mannosylation reactions in its Golgi, acquiring some of the α-mannose sugars and attaching it to its spike as a shield and then exocytosed from S. cerevisiae as a new CoV virus (assigned as CoV-C). CoV-C passes through the Golgi of pangolin cells where it is trimmed of some of its mannoses and then its glycoprotein is humanized (O-glycosylated) that led to emergence of another mutant of the virus (assigned as CoV-D). Last step in this interaction happens if a pangolin is co-infected by CoV-D and Mycobacterium avium strain 104. Both microbes have similar α-mannose epitopes and negatively charged surface due to presence of phosphate, so, they could share together to form a biofilm. Inside that biofilm, HBHA (Heparin-binding hemagglutinin) or extracellular DNA of Mycobacterium avium could be attached to CoV-D spikes at the S1/S2 fusion site (proteoglycan like site) introducing PRRAR sequence that triggered the furin cleavage at that site." Endocytosis, fermentation: The preferred carbon source for S. cerevisiae is glucose under both fermentative and non-fermentative conditions [47] ; however, when the supply of glucose is exhausted and oxygen is present, the cells can utilize other present sugars such as fructose or mannose [48] or even it can use nonfermentable carbon sources, such as lactate [49]. Spike glycoproteins of CoV viruses are highly mannosylated proteins varies from Man5GlcNAc2 to Man9GlcNAc2 glycans. Mannose can be utilized for energy production in S. cerevisiae and for mannosylation reactions in the yeast cell, including those involved in N-linked and O-linked glycosylation of yeast proteins and synthesis of the lipid anchor glycosylphosphatidylinositol [48,50]. So, the first step in microbial interaction is the endocytosis of (CoV-A) by Saccharomyces cerevisiae as a sugar.
Sugars endocytosis or uptake from the environment is controlled by a family of facilitative glucose transporters known as HXTs (hexose transporters) in Saccharomyces cerevisiae [51]. Once sugar get inside the cell, the concerted actions of glycolysis, citric acid cycle, and oxidative phosphorylation are used to generate a reservoir of adenonsine triphopshpate (ATP) [52]. In S. cerevisiae, utilization of mannose begins with its phosphorylation to mannose-6-phosphate (M6P) by hexokinase (Hxk1p or Hxk2p) or glucokinase (Glk1p). When yeast cells are grown on mannose as a carbon source, only HXK2 is highly expressed. M6P can either be converted to GDP-mannose to support mannosylation reactions in the cell or converted to fructose-6-phosphate (F6P) to fuel glycolysis (Fig. 2S) [53]. At this step, glycoprotein mannose sugar of CoV-A is fermented by Glk1p and HXK2 of S. cerevisiae leaving the virus naked from most of its glycan shield (assigned as CoV-B).
Phosphorylation: Glucose uptake/ATP energy balance in the cell of Saccharomyces cerevisiae is regulated by adenosine monophosphate (AMP) activated protein kinase called Snf1, that has been shown to phosphorylate and inhibit multiple members of a protein trafficking adaptor family, known as the αarrestins (Rod1, Rog3, and Csr2), which are responsible for the endocytosis. Phosphorylation of the αarrestins prevents α-arrestin-mediated endocytosis of glucose and other carbohydrate transporters [52,54]. Snf1 kinase could be the enzyme responsible for phosphorylation of Ser amino acids of the spike protein of SARS-CoV-2 which is phosphorylated parallel to α-arrestins after endocytosis of high amount of the virus and production of high level of ATP, (Fig. 3). Snf1 phosphorylate at Ser exclusively (i.e., not Thr) [54]. Protein phosphorylation is an extremely important mechanism of cellular regulation in Saccharomyces cerevisiae that expresses over 110 Ser/Thr protein kinases [55] that could also take part in Ser/Thr phosphorylation of SARS-CoV-2 spike protein.
N-glycan synthesis (α-mannosylation) in the Golgi apparatus of yeast: in contrast to mammals, yeast S. cerevisiae does not trim the ER-derived N-glycan but extends it either a core-type structure that contains just a few extra residues and is found on the intracellular glycoproteins, such as carboxypeptidase Y [56] or to a mannan structure that consists of a long branched polymer of approximately 200 mannoses attached to proteins [57]. GDP-mannose is utilized as a mannose donor in mannosylation in the Golgi with the help of several enzymes such as Och1p, (α-1, 6-mannosyltransferase) that adds one mannose residue to both types of N-glycan [57,58].
Coronaviruses have been previously reported to form virions by budding into the lumen of endoplasmic reticulum-Golgi intermediate compartments (ERGIC) [59][60][61] with the observations of hybrid-and complex-type glycans on virally derived material [62,63] .
In budding yeast Saccharomyces cerevisiae; Serine/threonine-linked glycans (O-glycans) of yeast species generally consist of α -1,2-and α -1,3-linked mannose residues [56]. Most of Ser/Thr sites at CoV-B are blocked through phosphorylation in the previous step, so O-glycosylation at these sites was not accomplished. N-glycosylation of spike protein of CoV-B was performed in a way that mimic the core-type structure that contains just a few extra residues of α-mannoses. In another words, S. cerevisiae considered CoV-B as its intracellular glycoproteins, such as carboxypeptidase Y and resulted in a new form of virus (assigned as CoV-C) (Fig. 3). CoV-C is now ready for exocytosis from the yeast cells to start a new modification pathway inside the mammalian or pangolin cells.

2-CoV spike O-glycosylation and N-glycoprotein humanization inside pangolin Golgi
In mammalian cells, some mannose residues in the core form, Man8GlcNAc2, are trimmed during protein transport from the ER to the Golgi apparatus, in which several different sugars such as Nacetylglucosamine, galactose, sialic acid, and/or fucose are added to achieve complexity [64]. And so; CoV spike glycoprotein (CoV-C) when enters the Golgi of pangolin cells will be subjected to subsequent events such as trimming of one mannose and then protein humanization that was carried out by fucosylation and glucosylation. Moreover, O-glycosylation of Spike protein of CoV-C could proceed also in this stage adding (O-GalNAc and O-GlcNAc) to S protein that was found to be O-GalNAcylated at Thr632, Thr673 and Thr678 and O-β-GlcNAcylated at Thr323, Thr632, Thr638, Ser686 [44]. These consequent events resulted in a new form of the virus assigned as CoV-D, (Fig. 4).

3-CoV-D hybridization with Mycobacterium avium
Mycobacterium avium is an ubiquitous environmental bacteria encountered in natural and urban water sources as well as soil [65] and is considered as opportunistic pathogen of humans [66][67][68][69], domestic and wild animals [70], poultry and fish [71].
Mycobacterium avium has been shown to produce a biofilm or a biofilm-like structure [72]. In M. avium biofilm matrix is associated with three components; a glycopeptidolipid (GPL) present on the cell wall [69], extracellular DNA (eDNA) that has been found to be a major component of bacterial biofilms produced by Mycobacterium avium subsp. hominissuis [73]. And as well, heparin-binding hemagglutinin (HBHA) adhesin; a bacterial cell surface-associated protein is one of the biofilm components [74].

Role of biofilm matrix of Mycobacterium avium in the interaction with CoV-D
At first, the bacterial cells detect required environmental signals including nutrients concentrations (glucose, indole, polyamines), inorganic molecules (iron, phosphate), pH, antimicrobials, temperature, oxygen concentration, osmolarity, and host derived signals (bile acids, hyrogen peroxide) [75][76][77]. Then the biofilm construction starts with reversible attachment of bacteria to a favorable surface according to the physicochemical and electrostatic interactions between the bacterial envelope itself and the substrate [75][76][77]. According to the previous parameters, CoV-D could be considered as an excellent substrate to which HBHA is expressed at the surface of a variety of mycobacterial species and promotes binding of the bacteria to each other and to host epithelial cells. It is the responsible adhesin for extra-pulmonary dissemination [79] and it is also considered as an important marker of latency [80].
No crystal structure of HBHA has been hitherto reported. Nevertheless, Small angle X-ray scattering and biophysical data provides a low resolution structure showed that HBHA has a dimeric structure that contains three functional domains: a transmembrane domain of 15-20 amino acids, the N-terminus of the protein; an α-helical coiled region which may be involved in protein oligomerization and a C-terminal region containing methylated lys-pro-ala-rich motifs [81]. It was reported that HBHA interact with host components such as cell surface sulfated glycoconjugates and proteoglycans [81,82] via the HBHA Cterminal domain by establishing electrostatic interactions with epithelial heparan sulphates [81,83].
HBHA protein sequence of Mycobacterium avium strain 104 was obtained from UniProt database  here that the C-terminal domain of M. Avium will bind to position 681 at the spike protein of CoV-D through one of the proline amino acids in this domain. Reviewing the literature, L-proline has been found to act as a weak agonist of the glycine and ionotropic glutamate receptors [84,85]. Proline residues reported also to have a unique roles in protein folding, structure, and function due to their conformational restriction, the absence of a hydrogen bond donor on the amide, and their special susceptibility to form a cis amide bond [86]. 2-Second scenario: Short polyproline motifs (domains that resemble the PXXP motif of human Piccolo protein) were detected in proteins from M. avium. This could make us speculate that these motifs could interact with SH3-containing proteins from the host to modulate its cell signaling pathways [88]. The yeast Saccharomyces cerevisiae was reported to have a proteome includes 29 SH3 domains distributed in 25 proteins [89]. A suggested mechanism is that SARS-CoV-1 through interaction with Saccharomyces cerevisiae created a SH3 like domain through molecular mimicry of yeast domains which enabled it later to interact with a proline rich motifs (PXXP) of M. avium that led to insertion of proline at that site.
Molecular mimicry is one of the strong strategies that enables a pathogen to colonize their hosts and enforce their replication and dissemination. Several viruses have gained the ability of interaction with host cell through protein short linear motifs (SLiMs) that mimic host SLiMs, thus facilitating their internalization and the manipulation of a wide range of cellular networks [90]. For example, the polyproline PxxPxR motifs; Nef and 5A protein (NS5A) found in the (HIV-1) and in the hepatitis C virus respectively. These motifs are able to interact with the SH3 domains of various host cellular proteins [90][91][92]. First scenario: HBHA expresses hemagglutination activity, a process correlated to interactions between HBHA molecules on the surface of mycobacteria. Hemagglutination has been correlated with the ability of the N-terminal region of HBHA to form multimers [93,94]. Proline residue at position 681 in CoV-D could act as a helical kink to which the amino acid (53-56) sequence; RRAR in H2 helix of the N-terminus of another HBHA is attached.

c) Introduction of LxxIxE-like motif (LDPLSE) at S1subunit of spike protein
This ensures the hypothesis of hybridization of SARS-CoV-1 like virus in pangolin with Mycobacterium avium strain 104 due to the presence of such motifs. As previously mentioned, LIDLQE motif in S2 subunit [46] reported in both SARS-CoV-2 and SARS-CoV-1 which ensure that the initial coronavirus that was manipulated inside pangolin was SARS-CoV-1 or a closely related species. LDPLSE is a unique motif present only in SARS-CoV-2 while it is absent in other viruses in the same family [46]. SARS-CoV-2 probably acquired this sequence through interaction with Mycobacterium avium. A conserved hypothetical protein of Mycobacterium avium strain 104 amino acid sequence was obtained from NCBI https://www.ncbi.nlm.nih.gov/protein/ABK66305.1?report=fasta , it contains the same sequence (

4-Furin cleavage
The spike protein of SARS-CoV-2 has a functional polybasic (furin) cleavage site (RRAR) at the S1-S2 boundary [44]. Furin is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells [97]. In general, PCSKs convert proproteins into their biologically active forms by cleaving them at specific target motifs made up of the basic amino acids lysine and arginine [98].
Upregulated furin A expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection [97]. This fact completes the whole picture of the hypothesis of recombination with M. Avium. If a pangolin co-infected with M. Avium and CoV, it will express high level of furin that will break the polybasic (RRAR) site at the S1-S2 boundary of the S protein in CoV-D and that resulted in the final form of this virus which is SARS-CoV-2. It is important here to note that furin enzyme was reported to be found at Malayan pangolin (Gene ID: 108385790) [99] and this fact ensure that the SARS-CoV-2 was transmitted to humans from pangolins not bats which lacks the furin enzyme. To can figures out this similarities or differences, a comparative study for the diseases caused by the three organisms; SARS-CoV-2, Mycobacterium avium and Saccharomyces cerevisiae was carried out. The comparative study showed similarity between the three disease and it includes the etiology (Table S1), symptoms (Table S2), clinical manifestations (Table S3), complications (Table S4), mode of transmission (Table S5), epidemiology (Table S6), herd susceptibility (Table S7), host cell interaction (Table S8), (Figs. 5S-7S), diagnosis (Table S9), (Fig. 8S) and treatment (Table S10)

1-COVID-19 is a contagious disease with a genetic predisposing factor
It is discussed earlier in this text that SARS-CoV-2 and Mycobacterium avium have similar antigenic epitopes (α-mannan glycans) (Figs. 1 and 3S). Mycobacterium avium infection has been reported from the Americans, Asian, and European patients [100] [101]. Other mycobacterial infections like Mycobacterium tuberculosis disease have the same epidemiology.
In the first half of the 19th century, tuberculosis peaked in Europe and one-quarter Europeans died of TB. Several studies referred to the role of human phenotypic traits, like lactose intolerance to be one of the reasons for which it was spread at Indo-European at that time [102]. Lactose intolerance (LI) is an interesting point to predict the ethnicity and hereditary factor for people who are more susceptible for SARS-CoV-2 infection. It is estimated that about 70% of the world population is affected by LI with great variation among ethnicities and races [103].
Recent publication discussed the increased prevalence of lactose mal absorption in the elderly due to clinically unapparent small intestinal bacterial overgrowth, rather than any subtle, age-related mucosal factors [104]. This could explain the prevalence of SARS-CoV-2 infection in elderly patients [105]. Beside the other risk factors such as the low immunity and chronic diseases, if we look to COVID-19 disease from LI notion, this could give an explanation for the prevalence of SARS-CoV-2 in older adult's population and in certain families with different ages as well as certain countries.
As reported by WHO [106], three ethnicities showed higher vulnerability for infection and were reported for the highest number of cases all over the world; Chinese, American and European (specially in Italy and Spain). Several studies reported LI in Chinese populations of all ages [107,108]. More recent study showed that 100% of Chinese showed lactase non-persistence [109]. LI also was reported among Europeans in several studies. For example; many studies discussed the LI among older Italian adults [110,111]. In Spain, IRISH Food Board reported that 34% of the Spanish population are lactose-intolerant [112].
Moreover, In the USA, there is LI in 70 to 80% of Whites of European or Scandinavian extraction, 30% of Mexicans, and 20% of African Americans [109]. While in countries like India showing a decline in the LI and it is only in some milk consuming nomads [109], where the rate of COVID-19 spread is limited.
Regarding Lactose intolerance, more information are needed for such potential association with SARS-CoV-2, no published data up to now about such possibilities and this hypothesis still need more clinical investigation and testing throughout the infected cases to confirm or counteract this likelihood correlation.
This hypothesis could also explain the reason for which blood group A was associated with a higher risk for acquiring COVID-19 [113] and as well the prevalence of vitamin D insufficiency in severe COVID-19 cases [114]. A group of researchers at the University of Toronto's Faculty of Medicine in Canada published a recent research stating that LI is associated with a lower plasma vitamin D concentration [115]. And as well, the correlation between blood type A and dairy products such as milk and cheese were discussed by many dietitians who described milk and other dairy products as the first diet enemy for people with blood type A. They owed the reason was due to many type A individuals have some degree of lactose intolerance, due to insufficient levels of lactase enzyme and then type A blood people creates antibodies to the indigestible sugar in dairy products [116]. Another predisposing factor for the spread of COVID-19 infection among the blood type A population is that the type As have a naturally high level of the stress hormone cortisol and produce more in response to stressful situations [116]. Cortisol is immunosuppressive in function, and elicits its immunosuppressive effects by downregulating key inflammatory transcription factors, NF-kB and AP-1, and upregulating the suppressor of cytokines [117].

Biofilm formation is a common phenomenon in bacteria and fungi and it was reported that
Mycobacterium avium [72] and Saccharomyces cerevisiae [72,118] have the ability to form a biofilm while a few or limited data are available on the ability of viruses to form a biofilm. HTLV-1 retrovirus is one of limited examples that was reported for its ability to form a biofilm like structures on the surface of infected cells [119]. The aggregates of HTLV-1 viruses embedded in a carbohydrate-rich structure containing cellsecreted extracellular matrix that may protect viruses from the immune system and enable them to spread efficiently from cell to cell. Extracellular matrix (ECM) components, including collagen, some specific heparan sulfate proteoglycans such as agrin, and several linker proteins such as galectin-3 and tetherin, were found to be enriched in these structures [119]. Comparing SARS-CoV-2 to HTLV-1 retrovirus, it is clear from the virus structure that SARS-CoV-2 genome is encased into a carbohydrate-rich adhesive extracellular 'cocoon', and also it has a proteoglycan like site (at S1/S2 fusion site in the spike glycoprotein) that provide a possibility for cell to cell transmission and formation of a biofilm like structure which enables its efficient and protected transfer between cells. Biofilm like structure could serve for the rapid transmission between individuals and inside the host cells, dissemination of the virus inside the host infecting several organs, relapse of infection, resistance to medication and longtime stability over a nonliving surface. Two recent publications about SARS-CoV-2 could support the hypothesis of the biofilm formation but this still need a laboratory confirmation. One study discussed the aerosol and surface longer stability of SARS-CoV-2 compared with SARS-CoV-1 [120]. The second study discussed the presence of SARS-CoV-2 in the urban water cycle and they owed the reason to many factors one of which is that the virus could colonize the bacterial biofilm [121].
The ability of the virus to form a biofilm is a good therapeutic target. The use of antibiofilm agents could be one of the therapeutic strategies applied for the treatment. Antibiofilm activity hypothesis could rationalize the French clinical therapeutic approach using Azithromycin [122] and explains its mechanism of action against SASR-CoV-2. Azithromycin has been reported for its antibiofilm activity through inhibiting the quorum sensing [123]. Several agents has been reported as antibiofilm agents with different mechanism of action such as quorum sensing inhibitors, biofilm matrix degrading enzymes, antimicrobial peptides, surfactants, free fatty acids, amino acids, nitric oxide generators, indole and its derivatives, anthranilate and metal chelators [124]. Accordingly, the current drug development trials are recommended to take into consideration the incorporation of antibiofilm agent into the supposed treatment formula.

4-The participation of Saccharomyces cerevisiae in the structure of SARS-CoV-2 explains some
of the abnormal clinical findings such as:

1-Kawasaki-like Syndrome among children
Since April 15, many European countries have reported the arrival of a surprisingly high number of children (3-17 years old) with symptoms similar to Kawasaki disease in intensive care of their hospitals [129] with no previous risk factors such as obesity or cardiac diseases. These cases showed symptoms of fever and abdominal pain or vomiting. Sometimes they had a very fleeting rash and presented with cardiac and circulatory failure. All the children needed ventilatory support, even if they did not have pulmonary disease. PCR test for SARS-CoV-2 among those cases was weak positive showing that they were infected 3-4 weeks before the appearance of symptoms [129].
The Saccharomyces cerevisiae infection among children was reported in many publications [130,131] nearly showed the same symptoms diagnosed in children with SARS-CoV-2. One case (3½-monthold male infant) of Saccharomyces cerevisiae infection showed symptoms of fever, watery diarrhea, erythematous macular skin rashes over the trunk and limbs, enlarged liver, few discrete palpable lymph nodes in the cervical region [132]. Other more severe cases developed sever reaction like septic shock [133], infective endocarditis, progressive renal, hepatic and pulmonary failure [134].

Kawasaki-like symptoms among children infected with SARS-CoV-2 is due to the presence of
Saccharomyces cerevisiae antigenic glycans over the surface of SARS-CoV-2 virus and this is one of points that could proof the hypothesis of the origin of COVID-19 disease generated in this article.

2-Skin hyper pigmentation of two Chinese physicians
A lot of newspapers and TV channels all over the world discussed the anomalous finding that A strange but more reasonable answer is the ability of the virus to synthesize melanin as a protective mechanism against ultraviolet irradiation for example. This still a theoretical explanation and it needs a laboratory investigation. Many fungal species produce melanin as a mechanism of resistance to environmental damage such as from ultraviolet radiation [135]. Studies with animal models demonstrate that melanin is a virulence factor responsible for dissemination in several fungal pathogens [135]. Previous studies reported some yeast species as a melanogenic yeast specially in the presence of DOPA but not the Saccharomyces cerevisiae [136,137] It is particularly important to investigate the ability of SARS-CoV-2 to form melanin especially after exposure to UV light which threatens infected medical team and if this comes true, COVID-19 patients should avoid prolonged exposure to sunlight.

5-The participation of Mycobacterium avium in the structure of SARS-CoV-2 explains the disseminated intravascular coagulation in COVID-19 patients
Blood from 20% to 30% of critically ill COVID-19 patients clotted in the tubes when nurses insert catheters for kidney dialysis and IV lines to draw blood as reported in many hospitals [138][139][140]. Several hospitalized patients have drastically elevated levels of a protein fragment called D-dimer, which is generated when a clot dissolve. High levels of D-dimer appear to be a powerful predictor of mortality in hospitalized patients infected with SARS-CoV-2 [141].
Since the year 2017, many publications reported the disseminated intravascular coagulation (DIC) as well as elevated D-dimer level in Mycobacterium avium complex infections [142][143][144][145][146]. Elevated D-dimer level has been also reported as a marker for infection with Mycobacterium tuberculosis [147]. It is reported that Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of Mycobacterium avium bind to fibrinogen (Fn) [148]. Assuming that SARS-CoV-2 has a portion like GAPDH acquired through microbial interaction, so it will be able also to bind to fibrinogen.
Fibrinogen is a glycoprotein complex that is converted enzymatically by thrombin to fibrin and then to a fibrin-based blood clot during tissue and vascular injury [149,150]. Thrombin is a as a serine protease enzyme catalyzing many coagulation-related reactions [151].
It is reported that SARS-CoV-2 has a serine protease enzymes [152] that could play the same role of thrombin when the virus bonded to fibrinogen and activates the conversion of fibrinogen to fibrin clots but this still need a laboratory and clinical investigation.

Conclusions, Recommendations and future perspectives
1) The human contact with pangolins through food or medical formulas should be halted and as well, yeast should not be applied as ingredient in the food formula provided to pangolins at captive breeding reservoirs.
2) Treatment: The bacterial and yeast share in the structure of SARS-CoV-2 could be a way to predict a treatment strategy. Up to date, some antibacterial drugs were tested against SARS-CoV-2 and showed activity such as Azithromycin, but no trial were done using the antifungal medications.
Depending on the hypothesis initiated in this article, it is recommended to start a clinical trials as well as a drug-drug interaction study with a triple therapy consist of antiviral (e.g. Remdesivir) + antibacterial (e.g. Aithromycin) + antifungal (e.g. fluorocytosine and/or Micafungin) medications.
The incorporation of antibiofilm agent in the drug formula is important to enhance the efficiency and reduce the resistance to treatment.

3) Vaccine or immunoprotecting:
The use of saccharomyces cerevisiae supplement as immunostimulant and protectant against SARS-CoV-2 as well as the development of yeast-based vaccines is highly recommended to be studied as a result of this hypothesis.

4) It is recommended to perform lactose intolerance test for all hospitalized patients to figures out this
risk factor which could be a useful diagnostic test to investigate the vulnerability to infection for people who have risk factors such as compromised immunity or chronic diseases or even for healthy person.

5) Laboratory investigation of the ability of virus to form a biofilm as well as the melanogenic activity
of SARS-CoV-2. This investigation is important for the therapeutic incorporation of antibiofilm agent in the medication and for the avoidance of patients to exposure to ultraviolet radiation.

Declaration -Ethical Approval and Consent to participate: No animal experiments or clinical trials were
performed.

SARS-CoV-2
It is a positive-stranded RNA virus with a crown-like appearance due to the presence of spike glycoproteins on the envelope. It belongs to Coronaviridae family which contains several CoVs that are classified into four types: Alpha, Beta, Delta, and Gamma coronaviruses. SARS-CoV-2 belongs to Beta-coronaviruses. [153].

Saccharomyces cerevisiae
Also known as "baker's yeast" or "brewer's yeast". It is a non-spore forming yeast that is widespread in nature and can be found on plants and fruit and in soil. It is mostly considered to be an occasional digestive commensal. S. cerevisiae is now included in some diet or health foods as well as some supplements or medications such as probiotic preparation containing Saccharomyces boulardii, a subtype of S. cerevisiae, used for the prevention and treatment of various diarrheal disorders. Since the 1990s, there have been a growing number of reports about the implication of S. cerevisiae as an etiologic agent of invasive infection association with a probiotic preparation of Saccharomyces boulardii (a subtype of S. cerevisiae) for treatment various diarrheal disorders [154]. S. cerevisiae is now considered an emerging opportunistic pathogen that has been related to a wide variety of infections, which range from vaginitis and cutaneous infections, to systemic bloodstream infections and infections of essential organs in immunocompromised and critically ill patients [155]. Several studies reported Saccharomyces cerevisiae associated pneumonia in immune-compromised patients [156][157][158].

Mycobacterium avium
It usually present as a complex with Mycobacterium intracellulare (MAC) because these species are difficult to differentiate [159]. M. avium complex is a nonmotile, slow-growing, non-spore-forming, gram-positive acid-fast bacillus, thermostable and can survive up to 49 о C [159].  avium subtype hominissuis appears to be gastrointestinal in origin. M. avium subspecies paratuberculosis was identified in ruminants as a causative agent for Johne disease [159,160].

SARS-CoV-2
The most common symptoms include fever, fatigue, and dry cough and shortness of breath. Less common symptoms include pharyngitis, headache, productive cough, GI symptoms and hemoptysis [161].

Mycobacterium avium
MAC infections symptoms depending on the site of the infection.
• The symptoms of pulmonary MAC infection include cough, weight loss, fever, fatigue, and night sweat [164]. • Disseminated MAC Symptoms include: Fever, sweating, weight loss, fatigue, diarrhea, abdominal pain, shortness of breath, anemia mastitis; pyomyositis and abscesses of the skin or brain [165,166]. • MAC lymphadenitis generally affects children who have normal immune systems. Symptoms usually only include swollen lymph nodes mainly on one side of the neck [165,166].

SARS-CoV-2
The clinical spectrum of COVID-19 varies from asymptomatic forms to clinical conditions characterized by respiratory failure that necessitates mechanical ventilation and support in an intensive care unit (ICU), to multiorgan and systemic manifestations in terms of sepsis, septic shock, and multiple organ dysfunction syndromes (MODS) [153].

Mycobacterium avium
MAC infections were classified to three types: • Pulmonary MAC infections (the most common type), that usually affect the lungs of elderly women and people who already have diseased lungs [164] , [166]. • Disseminated MAC infections , affect all organs throughout the body usually of AIDS patients [165]. • MAC associated lymphadenitis , characterized by neck lymph nodes swelling in young children who have normal immune systems [166,172].

Saccharomyces cerevisiae
Infectious endocarditis [176,177], infection of an aortic-bifemoral graft in some cases who developed aorto-enteric fistula and finally died of the infection [178].

Mycobacterium avium
• Anemia and weight loss are the most common complications reported with DMAC infection [179].
• High rates of morbidity and mortality were reported among HIV-infected patients with DMAC infections [179]. • Patients with more extensive disease have a 20% chance of relapse after treatment with anti-MAC drugs [179]. • Death could be a major complication for untreated patients with significant lung disease [179].
• Chronic Rhinosinusitis is also a known complication despite the use of antimicrobial drugs and sinus irrigation, 90% of patients had a persistent chronic symptoms such as headache, nasal congestion and decreased ability to taste or smell [180].

SARS-CoV-2
Currently the main transmission routes are respiratory droplets and contact transmission. Recent reports indicate that SARS-CoV-2 can be detected in the urine and stool of laboratory confirmed patients, implying a risk of fecal-oral transmission. There is still no evidence that SARS-CoV-2 can be transmitted from mother to baby during pregnancy or childbirth [181].

Saccharomyces cerevisiae
From abuse of probiotic preparations. It could also be transmitted in hospitals ICU (nosocomial acquisition) with catheters [154]. Transmission through hand was also reported [182,183].

Mycobacterium avium
MAC bacteria get into the body through inhalation of aerosol particles or ingestion of contaminated water [72,184]. Touching the same objects or having a close relationship with people who are sick from a MAC infection does not increase the infection. MAC infections was not considered as a contagious bacteria between persons [166].  [185].

Saccharomyces cerevisiae
The incidence of Saccharomyces fungemia varied from 1% to 3.6% in a retrospective study of 102 nosocomial cases of fungemia among patients at French teaching hospitals [154,186].

Mycobacterium avium
Mycobacterium avium complex is ubiquitous and has been reported from the Americas, Asia, and Europe [100,101].  [187].
Age: All ages could be infected but it is more common in elder people ≥ 60 years [188].

Risk factors:
Older adults and people of any age who have serious underlying medical conditions; chronic lung disease or moderate to severe asthma; serious heart conditions; immuno-compromised due to cancer treatment, smoking, bone marrow or organ transplantation, immune deficiencies, poorly controlled HIV or AIDS, and prolonged use of corticosteroids and other immune weakening medications; severe obesity (body mass index [BMI] ≥ 40); diabetes; chronic kidney disease undergoing dialysis and liver disease [189].

Mortality:
The mortality rate was 28%. The only factor that increased the mortality rate was older age [36].

Mycobacterium avium
Sex ratio: some studies revealed that women had a higher prevalence, up to 1.6-fold relative to men while other studies has shown a higher male prevalence (2:1) [159].

Risk factors:
Immune-compromised populations, such as patients with cystic fibrosis, chronic obstructive pulmonary disease, renal failure, transplant recipients with chronic corticosteroid use and TNF-α, and leukemia [68,69]. Some studies showed the importance of patient genetics in the infection vulnerability. A persons with Lady Windermere syndrome and patients with genetically determined defects of cell-mediated immunity such as abnormalities of the interleukin-12/interferon-γ axis, certain human leukocyte antigen alleles, cystic fibrosis transmembrane conductance regulator mutations, and polymorphisms of solute carrier 11A1 (or natural resistance-associated macrophage protein 1) and the vitamin D receptor are more susceptible to be infected by non-tuberculous mycobacterium [196].

Mortality:
A study measures the estimate of five-year all-cause mortality was 27% indicating a poor prognosis for patients [67].
• SARS CoV-2 is reported in a recent study to bind to CD209, a C-type lectin that binds to highmannose glycans on glycoproteins, has also been found to be as an alternative receptor for SARS-CoV [41], (Fig. 5S).

SARS-CoV-2
Radiology: Chest CT may show ground-glass opacities mainly involving the right lower lobes that may evolve into consolidation. Findings appear to peak at 10d of illness, resolution begins after day 14, Tree-in-bud signs, masses, cavitation, and calcifications were not observed (Fig. 8S) [161,187,211].

Laboratory testing:
RT-PCR assay is used to detect the viral loads in throat swab and sputum samples peaked at around 5-6 days after symptom onset [212].
Serologic testing: The IgM-IgG combined assay can be used for the rapid screening of SARS-CoV-2 carriers, symptomatic or asymptomatic [216].

Laboratory testing:
Blood cultures grew Saccharomyces cerevisiae that was identified by Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry [219]. Another study identified Saccharomyces by biochemical tests (API 20C AUX [Biomerieux] and Uni-Yeast-TEK [Remel]) and the presence of ascospores as well as sequence analysis of the 18S rRNA gene [178].
Laboratory testing: Diagnostic testing includes acid-fast bacillus (AFB) staining and cultures of sputum, blood, urine, stool and/or cutaneous lesions specimens depending on the type of infection [224].
In patients with DMAC, a complete blood count (CBC) often shows anemia, pancytopenia, leukopenia (lower CD4 cell counts), hypogammaglobulinemia, hypoalbuminemia, may be found. On liver function studies, patients with DMAC usually have elevated transaminase and alkaline phosphatase and lactate dehydrogenase (LDH) levels as well as elevated erythrocyte sedimentation rate (ESR) [224,225].
Serologic testing: An enzyme immunoassay (EIA) kit used in Japan has been used to detect serum IgA antibody to MAC-specific glycopeptidolipid core antigen [67,224,225].

SARS-CoV-2
UpToDate, there is no clinically proved effective antiviral treatment nor vaccine is currently available. The treatment is often symptomatic treatment including the oxygen therapy. In cases of respiratory failure, mechanical ventilation may be necessary for managing septic shock. Although no antiviral treatments have been approved, several approaches have been proposed such as lopinavir/ritonavir (400/100 mg every 12 hours), chloroquine (500 mg every 12 hours), and hydroxychloroquine (200 mg every 12 hours). Alpha-interferon (e.g., 5 million units by aerosol inhalation twice per day) is also used [153]. Preclinical studies suggested that remdesivir (GS5734)an inhibitor of RNA polymerase. could be effective for both prophylaxis and therapy of HCoVs infections [226]. Another drug that showed a promising activity against SARS-CoV-2 is favipiravir [227]. Azithromycin also showed a promising activity in combination with hydroxychloroquine [122].

Mycobacterium avium
Several medications are used in the treatment of Mycobacterium avium complex infections such as a macrolide, clofazimine, rifampin, rifabutin, ethambutol, fluoroquinolone, linezolid, and aminoglycosides. In severe cases including a fibrocavitary disease, a triple oral therapy consists of azithromycin, rifampin, and ethambutol is prescribed followed by injectable aminoglycoside. Inhaled amikacin can be used as well [159,229]. • Heparin-binding haemagglutinin binds to sulphated surface receptors such as heparine sulphate present on host cell and triggers mycobacterial dissemination which is important for its survival inside the host cell [204]. • Binding of lipoarabinomannan to CD209, a C-type lectin, stimulates Akt protein which phosphorylates Bad protein, and hence, the intrinsic apoptotic pathway is blocked [205]. • Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) binding to fibrinogen (Fn) on the host cell induces the uptake of iron which is to be acquired by the host cell for its own use [148]. • Adhesion of fibronectin-binding protein (FnBP) to fibronectin stimulates FAK/Src kinase which also leads to actin reorganization and also recruits tryptophan aspartate containing coat protein to early phagosome and prevents its fusion with lysosome. It also triggers calcium upregulation [206]. • Isocitrate Lyase binding with fibronectin or laminin has been shown. It mainly helps bacteria to survive under hypoxic conditions inside the host cell [207] . • Antigen-85 complex (85B and MPT51) binding with fibronectin/elastin induces interferon-γ (IFN-γ) which indicates that it also participates in host cell signalling mechanism [208]. • 19 kDa protein (maltose-binding protein) binds with mannose receptor and blocks human leukocyte antigen-antigen D-related (HLA-DR) protein present on major histocompatibility complex II (MHC-II) and leads to delayed antigen presentation [209].