Re iew
Pangolin Foo p in s: In ec i i y, Vi ulence and Long
COVID
Ge a d Kian-Meng Goh 1,*, A. Kei h Dunke 2, James A. Fos e 3,4 and Vladimi N. U e sky 5,6
Ci a ion: Las name, F.; Las name, F.;
Las name, F. Ti le. Biomolecules 2022,
12, x.
h ps://doi.o g/10.3390/xxxxx
Academic Edi o : Fi s name
Las name
Recei ed: da e
Accep ed: da e
Published: da e
Publishe ’s No e: MDPI s ay s
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claims in published maps and
ins i u ional a lia ions.
Copy igh : © 2022 by he au ho s.
Submi ed o possible open access
publica ion unde he e ms and
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(h ps://c ea i ecommons.o g/licen
ses/by/4.0/).
1Goh’s BioCompu ing, Singapo e 548957. Republic o Singapo e
2Cen e o Compu a ional Biology and Bioin o ma ics, Indiana Uni e si y School o Medicine,
Indianapolis, Indiana 46202, USA; [email p o ec ed]
3Depa men o Biological Sciences, Uni e si y o Idaho, Moscow, Idaho 83844, USA;
[email p o ec ed]
4Ins i u e o Bioin o ma ics and E olu iona y S udies, Uni e si y o Idaho, Moscow, Idaho
83844, USA
5Depa men o Molecula Medicine, Mo sani College o Medicine, Uni e si y o Sou h Flo ida,
Tampa, Flo ida 33612, USA; [email p o ec ed]
6Ins i u e o Biological Ins umen a ion, Russian Academy o Sciences, Pushchino, Moscow
egion, Russia
Co espondence: [email p o ec ed]
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Abs ac : This e iew ocuses on h ee closely ela ed models ha use p o ein in insic
diso de o link he N and M o in ec i i y, i ulence and, po en ially, long COVID.
While he models, Shell Diso de Models (SDMs), we e ini ially c ea ed using
compu a ional and empi ical molecula echniques based on p o ein in insic diso de ,
expe imen al and clinical da a we e con inuously used o e nemen and checked o
ep oducibili y and eliabili y o SDMs. In e es ingly, SDMs a e uniquely able o link
he po en ially majo cause o in ec i i y, i ulence and long COVID unde one cohe en
concep o p o ein in insic diso de . An example o his is SDMs' abili y o p o ide a
no el and cohe en explana ion o he di e ences in he i ulence and in ec i i y o
Omic on, SARS-CoV-1 and non-Omic on SARS-CoV-2. SARS-CoV-2 has been clinically
shown o induce much g ea e shedding o in ec ious pa icles in pa ien s. Cu iously, all
known SARS-CoV-2- ela ed i uses, excluding SARS-CoV-1, ha e an abno mally ha d
ou e shell (low M diso de ), which is associa ed wi h bu owing animals, such as
abbi s and pangolins. E idence o a unique molecula and e olu iona y ela ionship
(“pangolin oo p in s”) be ween pangolins and COVID-19 is examined. SDMs sugges
ha his ha d ou e shell is esponsible o he high in ec i i y o COVID-19 and ,
po en ially, long COVID, as he ha d M p o ides esis ance o he an imic obial
enzymes ound in he immune and espi a o y sys ems. The implica ions could p o ide
clues owa ds u he esea ch in ol ing long COVID and in ec i i y, including possible
ese oi s among mac ophages. This also explains clinical obse a ions o he
pe sis ence o he i us h oughou he body mon hs a e in ec ion. In he case o
i ulence, g ea e diso de in N con ibu es o mo e apid eplica ion by p o iding
mo e e cien p o ein-p o ein binding. As a esul s, N diso de co ela es wi h i al i e
and he e o e i ulence and, o some ex en , in ec i i y.
Keywo ds: co ona i us; COVID; in insic diso de ; memb ane; nucleocapsid;
nucleop o ein; Omic on; pangolin; shell; i ulence; long COVID; a enua ion; a ian ;
immune; pe o in; complemen sys em; mac ophage; ese oi ; A i cial In elligence;
AI; ha d shell; lysosome; uns uc u ed;NL63;spike;
Abb e ia ions:
COVID: Co ona i us Disease; CoV: Co ona i us; SARS: Se e e Acu e Respi a o y
Synd ome; HCoV: Human co ona i us. SDM: Shell Diso de Model;, PONDR®:-VLXT:
P edic o o Na u al Diso de ed Regions using VLXT; PID: Pe cen age o In insic
Diso de (numbe o diso de ed esidues di ided by he o al numbe o esidues);
Pang2017: SARS-CoV-2 ela ed pangolin-CoV isola ed in 2017; Pang2019: Pangolin-CoV
isola ed in 2019, N: Nucleocapsid p o ein, M Memb ane p o ein; S: Spike p o ein;
SARS-CoV-2, BANAL: SARS-CoV-2 ela ed Ba -CoVs ound in Laos, NL63: A common
HCoV; RaTG13: A SARS-CoV-2 ela ed ba -CoV disco e ed in Yunnan; AI: A i cial
in elligence; EBOV: Ebola i us; NiV: Nipah i us; DENV: Dengue i us; HIV: Human
immunode ciency i us; YFV: Yellow e e i us; ZIKV: Zika i us;
1. In oduc ion
1.1 O e iew
This e iew ocuses on he h ee closely ela ed models as ela ed o COVID-19
(Co ona i us Disease 2019) [1-3]. The models use p o ein in insic diso de o link he
N and M o in ec i i y, i ulence and, po en ially, long COVID. While he h ee models,
Shell Diso de Models (SDMs) [4], we e compu a ionally c ea ed using empi ical
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molecula echniques based on p o ein in insic diso de , expe imen al and clinical da a
will be examined o he ep oducibili y and eliabili y o SDMs. In e es ingly, SDMs
a e uniquely able o link he po en ially majo cause o in ec i i y, i ulence and long
COVID unde one cohe en concep o p o ein in insic diso de . An example o his is
SDMs Fo ins ance, i is able o p o ide a no el and cohe en explana ion o he
di e ences in i ulence and in ec i i y be ween SARS-CoV-1 and SARS-CoV-2, which
has been clinically shown o induce much g ea e shedding o in ec ious pa icles in
pa ien s. Cu iously, all known SARS-CoV-2- ela ed i uses, excluding SARS-CoV-1,
ha e an abno mally ha d ou e shell (low M diso de ), which is associa ed wi h
bu owing animals, such as abbi s and pangolins. E idence o a unique molecula and
e olu iona y ela ionship (“pangolin oo p in s”) be ween pangolins and COVID-19 is
examined. SDMs sugges s ha his ha d ou e shell is esponsible o he high
in ec i i y o COVID-19 and, po en ially, long COVID, as he ha d M p o ides
esis ance o he an imic obial enzymes ound in he immune and espi a o y sys ems.
The implica ions could p o ide clues owa ds u he esea ch in ol ing long COVID
and in ec i i y, including he sea ch o possible ese oi s among mac ophages. This
also explains clinical obse a ions o he pe sis ence o he i us h oughou he body
mon hs a e in ec ion. In he case o i ulence, g ea e diso de in N con ibu es o
mo e apid eplica ion by p o iding mo e e cien p o ein-p o ein binding. As a esul s,
N diso de co ela es wi h i al i e and he e o e i ulence and, o a limi ed ex en ,
in ec i i y. As S (spike p o ein) is cu en ly ye o be pa o SDMs, S is men ioned
spa ingly as pa o a discussion in ol ing he po en ials and limi a ions o SDMs. I
should also be eminded ha while he e a e cu en e iew and esea ch pape s ha
a emp o link a ious i al p o eins o i ulence o in ec i i y [5-8], we will a emp o
show ha he app oach using p o ein in insic diso de p o ides a mo e cohe en
concep ha links i ulence and in ec i i y ia M and N. S could also be la e added o
he amewo k a some poin .
1.2 Pangolin Foo p in s: Enigmas o COVID-19 Rela ed Vi uses
I has been mo e han 4 yea s since he s ou b eak o COVID-19 [1-3]. E en
as we a e jus beginning o unde s and wha was p e iously unclea abou i ,
he e is s ill much o be unco e ed and esol ed. Fo ins ance, why is SARS-
CoV-2 highly con agious in con as o 2003 SARS-CoV (SARS-CoV-1, SARS:
Se e e Acu e Respi a o y Synd ome, CoV: Co ona i us)? The o al numbe o
people in ec ed hus a is mo e han 700 million [3], whe eas he e we e only
abou 10,000 cases in he 2002-3 SARS ou b eak [4]. Why is SARS-Co-2 less
i ulen and much mo e in ec ious han SARS-CoV-1? Wha a e he s uc u al
di e ences esponsible o he di e ences? Wha is he cause o long COVID?
Ea ly in he ou b eak i was belie ed ha he eason o he ex ao dina y
con agiousness o COVID-19 lies solely on he Spike (S) p o ein [9-12]. As mo e
compu a ional, clinical, and expe imen al da a became a ailable, he
ep oducibili y o such a hypo hesis can be inc easingly ques ioned e en
hough he e a e ew cu en pape s ha examine his issue especially
pe aining links be ween in ec i i y and i ulence in a cohe en manne , which
we belie ed is bes app oached using he concep o p o ein in insic diso de .
This pape e-examines he oles o wo highly impo an , hough less-
esea ched, p o eins: M and N, while keeping he unc ions o S in mind.
The amewo k o many o he s udies we e es ablished using an AI ool
o s udy he sequence o he M and N p o eins. One impo an bu peculia
disco e y using he p o ein diso de AI ool, PONDR®-VLXT [13-17], is ha
all SARS-CoV-2- ela ed i uses, no jus SARS-CoV-2, ha e among he ha des
ou e shells (low M diso de ) known wi hin he CoV amily [18-21]. I is
belie ed ha i is his anomaly pe aining o M ha is p ima ily esponsible o
he high con agiousness o COVID-19, since a ha de M p o ides g ea e
esis ance o he i us agains he la ge a ay o an imic obial enzymes p esen
in he sali a and mucus [21-28] and, hus, making i mo e likely o he hos o
shed much mo e in ec ious pa icles [29]. N, on he o he hand, could help
modula e he in ec i i y and i ulence as g ea e N diso de allows o mo e
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e cien p o ein-p o ein/RNA/lipid binding [4,16,30-33] ha could lead o
mo e apid eplica ion o he i us, especially in i al o gans [34-40].
A e ospec i e sea ch a e he ini ial COVID-19 ou b eak yielded a ba -
CoV sample (RaTG13) ob ained om a Yunnan ca e in 2013 ha had a 96.4%
gene ic iden i y wi h SARS-CoV-2 [4-43]. Fu he mo e, wo se s o pangolin-
CoVs ha we e ob ained om pangolins con sca ed by cus oms in Guangxi
(GX) and Guangdong (GD) p o inces du ing 2017-18 (Pang2017, Pang2018)
and 2019 (Pang2019) pe iods espec i ely ha e abou 90% gene ic p oximi y o
SARS-CoV-2 [44-48]. La e , pangolin samples ob ained in Vie nam showed
simila esul s [49]. Likewise, a se ies o COVID-19 ela ed ba -CoVs (BANAL)
we e ound in Laos [50-51]. In ac , one o he samples, BANAL-52, had an e en
g ea e gene ic p oximi y (96.8%) o SARS-CoV-2 han ha o RaTG13 o SARS-
CoV-2.
A sea ch o simila ly ha d M yielded CoVs associa ed wi h bu owing
animals such abbi s. This enigma ic associa ion explains he ue in ima e
ela ionship be ween all COVID-19 ela ed i uses and pangolin-CoVs since
pangolins a e also bu owing animals [4,18-20,52-54]. The ha d ou e shell (M)
is necessa y since he i us has o be able o su i e longe in bu ied eces
be o e u he ansmission.
While mos scien is s belie e ha COVID-19 is he esul o a zoono ic
spillo e , a be e unde s anding o he e olu ion helps. The knowledge o N
and M p o eins can make he unde s anding o his e olu ion mo e comple e
as i b idges he ela ionship be ween SARS-CoV-2 and pangolin-CoV. The
ela ionship as e idenced by he pangolin molecula “ oo p in s” can accoun
o many o he beha io s o COVID-19. The implica ions o his ela ionship
will be e- isi ed in g ea e de ail la e .
.
One odd cha ac e is ic ound in all SARS-CoV-2- ela ed i uses and hei
a ian s ound hus a is he unusually ha d ou e shell (low M diso de ) ha
is ypically ound only in CoV associa ed wi h a bu owing animal. This
hallma k is one o he “pangolin oo p in s” ound in all COVID-19 ela ed
i uses disco e ed so a . The o he se o oo p in s in ol e a enua ions
a ising o lesse diso de in N ound in Pang2017 and, la e , in he a ian
Omic on [18-20,52-56]. The ha de inne shell (N), especially in Pang2017, may
be also a e ec ion o N p o ec ing he i al RNA in bu ied eces, jus like M
[52,55].
E idence o pangolin oo p in s o e s clues ha a e beginning o unco e
many o he mys e ies o COVID-19 ha a e s ill haun ing us. These include
ques ions such as: why is SARS-CoV-2 much mo e in ec ious han SARS-CoV-
1? Why is COVID-19 is highly in ec ious o his day? Why is SARS-CoV-2 less
i ulen han SARS-CoV-1? Why is Wuhan-Hu-1 much mo e i ulen han
Omic on? Wha is he ac ual s uc u al cause o long COVID? Many o hese
ques ions ha e emained unanswe ed, bu he s udy o he M and N p o eins is
now beginning o p o ide some answe s om one in eg a ed concep , namely
he pangolin oo p in s. We should no be a all su p ised by he explana o y
p owess o he N and M p o eins, as hey a e he mos abundan p o eins
ound in he cell and i ion, espec i ely.
1.3 Long COVID
Ye ano he majo mys e y in COVID-19 is he p esence o long COVID among
many pa ien s, i.e. he pe sis ence o symp oms long a e eco e y [57-58] . I
should also be no ed ha S, unlike M, is unable o accoun o he pe sis ence
o he i us e en mon hs a e he ini ial in ec ion as obse ed by clinical
s udies. E en as p og ess has been made in esea ch o explain he mechanism
and cause o long COVID, long COVID has emained by and la ge a mys e y
[59-64]. In his pape , we will e iew an al e na i e explana ion ha has
al eady been p e iously men ioned. This explana ion in ol es he unusually
ha d M ound in SARS-CoV-2 ha allows he i us o esis immune enzymes
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and enables he i us o hide in mac ophages. The abno mally ha d ou e shell,
M, ha p o ec s he i ion om he onslaugh o he an imic obial enzymes
ound in sali a and mucus [19-28,40,52], is also likely o educe he chances o
he elimina ion o i al pa icles ia ac ions o he immune sys em. We will
examine li e a u e pe aining o cu en knowledge o immunology ha could
p o ide a po en ial amewo k o he mechanism o long COVID caused by
ha d M . SDMs will be used o gain u he insigh o he po en ial mechanisms
in ol ed.
The abno mally ha d ou e shell, M, ha p o ec s he i ion om he
onslaugh o he an imic obial enzymes ound in sali a and mucus [19-
28,40,52], is also likely o educe he chances o he elimina ion o i al pa icles
ia ac ions o he immune sys em. Upon examina ion o immunological
p inciples, Shell-Diso de Models (SDMs) e en go on o pinpoin he exac
immunological mechanisms ha a e likely o be hinde ed.
2. The Shell Diso de Models (SDMs): Th ee Closely Rela ed Models
2.1 Th ee Closely In e ela ed Models
Th ee closely ela ed models we e de eloped using he concep o p o ein
in insic diso de ia AI ools. P o ein in insic diso de e e s o he lack o
s uc u e in an en i e o pa o a p o ein. Diso de is known o ha e a ious
i m p o a n u n c i o n s e s p e c i a l l y i n p o e i n -
p o ein/RNA/DNA/lipid/ca bohyd a e binding, and a ious ools ha e been
de eloped o ecognize diso de egions and diso de ed p o eins. Among he
s de eloped is PONDR®-VLXT, which in ol es he use o neu al ne wo k
AI o ecognize diso de ed esidues gi en he sequence inpu [13-17]. PONDR®-
VLXT has been shown o be a highly app op ia e ool pa icula ly when i
in ol es i al p o eins o a la ge a ie y o i uses including Ebola i us
(EBOV), Dengue i us (DENV), Nipah i us, and HIV [4,18-20,34-40,52,-56,65-
67]. PONDR®-VLXT is especially sui ed o he s udy o i al s uc u al
p o eins as i is highly sensi i e in he de ec ion o diso de in s uc u ed
p o eins [16]. The s SDM was ini ia ed be o e 2008 when PONDR®-VLXT
was used o examine he shell p o eins o a a ie y o i uses. A use ul numbe
used is pe cen age o in insic diso de (PID), which is de ned as he numbe
o diso de ed esidues p edic ed di ided by he o al numbe o esidues in a
p o ein chain [4,65]. A diso de ed esidue is p edic ed o be diso de ed i i s
VLXT sco e is 0.5 o abo e. Upon compa ison o he shell diso de o a ai ly
la ge numbe o i uses, i became ob ious ha he ou e shell o HIV,
especially HIV-1, has an abno mally high a e age diso de in i s ou e shells. I
was also disco e ed ha HSV and HCV sha e his simila cha ac e is ic, e en
hough i is no as p onounced as in HIV-1 [418-20,34-40,65-67]. Since e y ew
o he i uses, i any, ha e his cha ac e is ic, i seems o ha e o do wi h he
abili y o he i uses o e ade he immune sys em, and esul s in he absence o
e ec i e accines o he men ioned i uses. This SDM was labeled “Vi al
Shapeshi ing” [4] and became he pa en model o wo o he closely ela ed
SDM, as seen in Figu e 1. Gi en he beha io s o HIV, HCV, and HSV, highe
diso de a he ou e shell can also be associa ed wi h he abili y o pene a e
ha d- o- each places such as he b ain and placen a, as in he case o he Zika
i us [34,35]. P o ein diso de p o ides o g ea e e ciency in p o ein-
p o ein/DNA/RNA/lipid/glycop o ein binding [16.30-33,55,58] .
Table 1. The Th ee Shell Diso de Models (SDMs). SDMs we e de eloped using
p inciples o p o ein in insic diso de applied o i al shell p o eins.
Yea o Fi s Shell Diso de De ails
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Publica ion Model
2008
Vi al
Shapeshi e
Model (Pa en )
Diso de o shell p o eins was measu ed o a wide
a ie y o i uses. Only e y ew i uses ha e been
ound o ha e unusually high diso de a he ou e shell
(HIV-1, HCV, and HSV). The e is no e ec i e accine ye
ound o he h ee i uses.
2012
CoV
T ansmission
SDM
Links be ween modes o ansmission ( ecal-o al and
espi a o y) and N-M diso de we e ound.
2015
Vi ulence-
Inne Shell
Diso de
Model
S ong co ela ion be ween inne shell diso de and
i ulence o a wide a ie y o i uses including DENV,
EBOV, NiV, and SARS-CoV-1/2.
Ano he model was de eloped and published in 2015 when i was disco e ed
ha he e is a s ong co ela ion be ween DENV i ulence and diso de a he
inne shell p o ein [34,35]. Simila co ela ions we e ound in a la ge a ie y o
o he i uses including NIV, CoVs, and EBOV [4,34-40,52-56,65-67]. The
p esence o such co ela ions has o do wi h he ac ha he inne shells a e
o en associa ed wi h eplica ion in many i uses, and because diso de
p o i d e s o g e a e e c i e n c y i n p o e i n -
p o ein/RNA/DNA/lipid/glycop o ein binding [16,30-33]. These o m he
basis o he Vi ulence-Inne Shell-Diso de Model (Table 1).
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Figu e 1. Vi ulence-Inne SDM (Shell Diso de Model). A. Shells PID o DENV and SARS-CoV-2. Inne shell o DENV
has been ound o be co ela ed o i ulence ( =0.95) [34,35]. B. Co ela ion be ween he SARS- ela ed i uses and N
PID. The co ela ion is based on es ima ed CFRs o SARS-CoV-1/2 and Omic on. No e: DENV and SARS-CoV-1/2 shells
a e no co ela ed. They a e jus placed oge he o illus a i e pu pose only.
The hi d SDM was s published in 2012 [53] be o e MERS-CoV was
disco e ed in 2013 [55]. The model di ided CoVs in o h ee g oups, labeled A-
C, ha can be seen in Figu e 2, wi h g oup D added du ing he COVID-19
pandemic [18-20,40,53,55,56]. G oup D was no ecognized in he ini ial model
because he e we e e y ew CoVs ha in ol ed bu owing animals such as
abbi s and pangolins a ha ime [4,18-16,52,53]. Be o e he COVID-19
pandemic, nea ly all CoVs in a ailable ou da abase ha e M PIDs o a leas 8%
(See Figu e 2 and Supplemen a y Table). The excep ions ha ha e M PIDs
lowe han 8% a e CoVs associa ed wi h bu owing animals. I was du ing he
pandemic ha i was disco e ed ha all SARS-CoV-2- ela ed i uses ha e M
PIDs lowe han 7%(4-6.3%) (See Figu e 2 and Table 2). While he s a is ical
di e ences seem small, in eali y, i is ac ually no any hing small o i ial as
we will see, la e , ha M is he mos abundan p o ein ha encases he en i e
i ion. E en small changes will a ec he igidi y o he en i e shell. This CoV-
T ansmission SDM in okes he same molecula p inciple ha he Vi ulence-
Inne SDM uses, which in ol es g ea e diso de a he inne shell ha
p o ides g ea e e ciency in i al eplica ion. Howe e , he CoV-
T ansmission SDM ex ends he p inciple o he le els o ecal-o al and
espi a o y ansmission po en ials, by showing ha espi a o y ansmission is
iable only when su cien copies o he i us a e shed nasally. This esul s in
N being adequa ely diso de ed. Mul i a ia e analysis ha e ound s ong
co ela ion be ween modes o ansmission and le els o M/N PIDs wi h
s a is ical signi cance (Mul i a ia e analysis: p < 0.001, ~0,8, N = 32, see Suppl.
Table o u he in o ma ion).
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Figu e 2. CoV T ansmission Shell Diso de Model (SDM) CoVs. In g oups A-C, he
le els o espi a o y/ ecal-o al ansmission a e hea ily dependen on N PID, whe eas
hose in g oup D ha e unusually low M PIDs (M diso de ) ha a e usually associa ed
wi h bu owing animals such as pangolins. G oup D includes all COVID-19- ela ed
i uses. (Mul i a ia e analysis: p < 0.001, ~0,8, N = 32, see Suppl. Table o u he
in o ma ion)
The CoV-T ansmission SDM ca ego ies SARS-CoV-1 in g oup B consis o
CoVs wi h in e media e ecal-o al and espi a o y ansmission po en ials.
Upon he publica ion o he o iginal pape , he MERS-CoV ou b eak ook place
in 2012-13 when he SDM had o place MERS-CoV in g oup C, in which he
CoVs ha e highe and lowe ecal-o al and espi a o y ansmission po en ials
espec i ely [54]. This p edic ion has been ep oduced clinically and
expe imen ally [4]. I is also known ha MERS-CoV has long been en enched
among camels, especially a med camels, whe e i sp eads easily by ecal-o al
means.
3. Pangolin Foo p in s: Applying SDMs o SARS-CoV-2
3.1.. The Fi s Sign o a Pangolin Foo p in : Abno mally Ha d Shell in All SARS-
CoV-2-Rela ed Vi uses
When he CoV-T ansmission SDM was s de eloped and implemen ed
in 2012-13, s ong co ela ion was seen be ween N diso de (N PID) and he
modes o ansmission [4,53,54]. Howe e , s a is ical calcula ions de ec ed a
small co ela ion be ween M diso de (M PID) and modes o ansmission. PID
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is de ned as he numbe o diso de ed esidues di ided by he o al numbe o
esidues in he p o ein. A ha ime, i was no unde s ood why such a
co ela ion exis ed. I was no un il he a i al o he COVID-19 pandemic ha
came wi h a o en o da a ha hings began o all in place. The CoV-
T ansmission SDM was applied o he Wuhan-Hu-1 as soon as he M and N
p o eins became a ailable, wi h Wuhan-Hu-1 ha ing N and M PIDs o 48.2%
and 5.9% espec i ely [14,40,52-55]. Using he o iginal model, SDM placed
SARS-CoV-2 in g oup B, which is he same g oup ha SARS-CoV-1 is in.
SDM did de ec some hing highly unusual abou his i us ha was
seldom seen in ou cu a ed da abase o known CoVs: he ou e shell o SARS-
CoV-2 is abno mally ha d, i.e. has a low M PID [18-20]. As mo e da a became
a ailable, i became clea ha he odd ha d ou e shell was no jus some hing
ha pe ains only o SARS-CoV-2 bu o all COVID-19- ela ed i uses, as seen
in Table 2 and Figu e 2. We can see ha he ha d M (low M PID < 7%, Table 2)
ex ends o all SARS-CoV-2- ela ed i uses, including pangolin-CoVs and ba -
CoVs such as RaTG13 and he Lao ian ba -CoV (BANAL). De ails ound in he
ables can also be ound in p e ious pape s [52,55-56] and he p o ein
sequences we e ob ained ei he om UniP o [69] o NCBI-P o ein [70], and
he PONDR®-VLXT sco es we e ob ained by inpu ing he sequences in o
PONDR®-VLXT [13-17]. The PIDs (pe cen ages o in insic diso de ) we e
calcula ed as he numbe o diso de ed esidues di ided by he o al numbe o
p o ein in a p o ein chain [4,65-67].
Table 2. N/M PIDs and Sequence Simila i ies o COVID-19 Rela ed Vi uses CoVs wi h
SARS-CoV-1 and Non-SARS-CoV-2- ela ed Ba -CoVs as e e ences. I should be no ed
ha BA1 was he ini ial Omic on. A leas wo Del a sub a ian s ha e been de ec ed by
SDMs.
Co ona i us Sequence
Simila i y
M (%)
M PID
(%)
Accession:
UniP o (U);
GenBank (G)
Sequence
Simila i y
N (%)
N PID
(%)
Accession
UniP o (U);
GenBank(G)
SARS-CoV-1
Ci e -SARS-CoV
90.5
90.1
8.6
8.6
P59596(U)
Q3ZTE9(U)
90.5
90.01
50.2
49.1
P59595(U)
Q3ZTE4(U)
Lao ian Ba -CoV
[Banal-52]
[Banal-103]
[Banal-236]
-
98,7
98.7
99.1
6.0+0.2
6.3
5.9
4.1
-
UAY13220.1
UAY13232.1
UAY13256.1
-
99.3
99.1
99.3
48.3+0.2
48.2
48.5
48.5
-
UAY13225.1
UAY13257.1
UAY1326.1
Pangolin-CoV
2019
2018
2017
-
98.2
97.7
98.2
5.6+0.9
6.3
4.5
5.9
-
QIG55948(G)
QIQ54051(G)
QIA48617(G)
-
98
93.8
94
93.32
46.6+1.6
48.7
46.3
44.9
46.5
-
QIG55953(G)
QIQ54056(G)
QIA48630(G)
QIA48656(G)
SARS-CoV-2
Wuhan-Hu-1
Del a
Del a1
Del a2
Omic on
Omic on BA1
Omic on XBB
100
99.1
99.1
-
98.7
99.1
5.9
5.9+0.01
5.9
5.9
5.7+0.4
5.4
YP009724393(G)
QUX81285(G)
QUX81285(G)
-
UFO59282(G)
WBI50320(G)
100
99.1
-
98.6
48.2
47.1+0.5
46.8
47.5
44.5+0.4
44.8
44.2
YP009724397(G)
QYM89997(G)
QYM89845(G)
-
UFO692871(G)
WIL50325
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3.7. Phylogene ic T ees Using M Re eal a Mo e In ima e Rela ionship Be ween
Pangolin-CoVs and SARS-CoV-2: Ano he Sign o a Pangolin Foo p in
We ha e seen ha a ha d M is he hallma k o all hus-known COVID-19
i uses. Being ha d o o de ed usually en ails a mo e conse ed p o ein. Based
on his, M is e y likely o be highly conse ed among all COVID-19 ela ed
i uses. Such a ea u e makes M mo e ideal o phylogene ic s udies, as i is
known ha ecombina ions could cause g oss e o s in phylogene ic
calcula ions. In e es ingly, phylogene ic calcula ions yielded esul s ha a e
di e en om hose using he en i e genome o o he p o eins [41-48,52,55].
Figu e 5A,C uses M o show ha pangolin-CoVs ha e a much close
ela ionship o SARS-CoV-2, no seen when o he p o ein such N (Figu e 5B) o
en i e genome is used.
Figu e 5A is di e en om Figu e 5C as hey in ol e sligh ly di e en
algo i hms. In any case, bo h show ha pangolin-CoVs ha e in ima e
ela ionships wi h SARS-CoV-2. I is in e es ing o no e ha he wo i uses
ha ha e he g ea es sequence simila i ies o SAR-CoV-2 a e ba -CoVs,
RaTG13 and BANAL-52, wi h 96.1% and 96.8% espec i ely [51-52]. Wha is
in iguing, howe e , is ha in Figu e 3A, i can be seen ha BANAL-52 and
Pang2019 ha e he simila ly closes ela ionship o SARS-CoV-2, in con as o
RaTG13. How can his be when RaTG13 has a 96.1% sequence iden i y o
SARS-CoV-2, compa ed o abou 90% o Pang2019? Sequence iden i y does
no o e he ull pic u e because o he possibili y o ecombina ion occu ing.
Fu he mo e, phylogene ic gene ic algo i hms end o make mis akes when
ecombina ion had aken place [94]. The abno mally ha d M ound in all
COVID-19- ela ed i uses en ails a s uc u al and gene ic conse a ion, which
means ha he likelihood o any ecombina ion ha ing aken place is much
lowe . The e o e, we belie e ha Figu e 5A,C p esen s he mos accu a e
phylogene ic s udy by a oiding he chances o ecombina ion.
Ye ano he odd ea u e can be seen in Figu e 5C, whe e Omic on is mo e
closely ela ed o he pangolin-CoVs han o he o he a ian s [40,52,55]. While
his may seem odd, we know ha Omic on i sel is sh ouded in mys e y. When
Omic on was i s sequenced, i was ound o ha e mu a ions ha a e unlike any o he
a ian s. The ques ion ha quickly a ises is: Whe e has Omic on been hiding all along?
[95]. Why didn' he medical and scien i ic communi y no ice i i i was in he human
popula ion? The e we e a ew sugges ions. A ew scien is s sugges ed ha he i us
was hiding in a small g oup o immunocomp omised people such HIV o cance
pa ien s [96]. O he s ha e sugges ed ha he i us had been hiding in an animal such as
a o pangolin. One pape has sugges ed ha Omic on had been hiding among mice
based on he mu a ions o i s S and mouse ACE-2 [96]. Diso de s udies on Omic on
do sugges ha i could be hiding in a bu owing animal such as pangolin as he i s
Omic on a ian BA1 had an e en ha de M han o he SARS-CoV-2 a ian s (M
PIDs: 5.4% Vs 5.8%) [55]. The idea ha Omic on had been hiding in mice ac ually
does no con adic he sugges ion in he p e ious s a emen since mice a e also
bu owing animals. A complica ion a ises, howe e , when we examine he e olu ion o
mice and a s. While a s and mice dwell in bu ows in he coun yside, a s and mice
in he u ban se ings ha e e ol ed o li e in he homes o human (Schmid -Holmes e
al) [97]. The e o e, depending on he species, hey could ha e cha ac e is ics o bo h
bu owing and non-bu owing animals. The phylogene ic ee (Figu e 5C) ha shows
an unusually close ela ionship be ween Omic on and pangolin-CoVs, in con as o he
o he a ian s, adds an impo an clue owa ds sol ing he puzzle. The phylogene ic
ee is sugges ing ha i was li e ally hiding wi hin a popula ion o a
bu owing animal such as pangolins.
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Figu e 5. A CoV Phylogene ic S udy T ees Using M. A. Phylogene ic s udy o CoVs using M ia CLUSTAL OMEGA [89].
B. Phylogene ic s udy o CoVs using N ia CLUSTAL OMEGA [98-99] . C. Phylogene ic s udy o COVID-19- ela ed
i uses using M ( ia CLUSTALW [100]). Vi uses ela ed o SARS-CoV-2/1 a e shaded in blue in (A) and (B).
4. The Shell Diso de Models and Rep oducibili y: M and N P o eins
4.1. The P oblem wi h he S P o ein: Limi a ions and Po en ials
When COVID-19 s s uck in Wuhan i sp ead globally wi h a e oci y
no seen since SAR-CoV-1, despi e e o s o con ol i . Almos immedia ely,
many scien is s began o ze o in on S as he p o ein esponsible o i s high
in ec i i y [9-11]. A compu a ional model sugges ed ha he SARS-CoV-2 S
binds mo e e cien ly o he ACE-2 ecep o han SARS-CoV-1 [9-11].
Fu he mo e, a speci c polybasic sequence known as he u in clea age (FCS)
can only be ound in SARS-CoV-2 bu no in he COVID-19- ela ed ba -CoVs
and pangolin-CoV and SARS-CoV-1 [101-103]. Many scien is s hailed S, as well
as FCS, as he holy g ail o COVID-19 ansmissibili y and i ulence
knowledge [102-103].
The S p o ein has been widely s udied [104] since i plays impo an oles
wi h many implica ions. I is in ima ely in ol ed in i al en y, which p esen s
an oppo uni y o he disco e y o d ugs and accines ha block he
a achmen o he i us o he hos cells [104]. Fu he mo e, being a su ace
p o ein, i is being widely s udied o he way i s mu a ions help e ade he hos
immune sys em. I is he e o e no di cul o see why S is he mos s udied
CoV p o ein. S is, howe e , so in ensi ely s udied ha i is easy o ge he
imp ession ha i is he only p o ein ha ma e s, o ha no o he CoV p o ein
exis s. This pa adigm is, o cou se, nonsensical as i iola es a basic ene o
biochemis y: each p o ein plays indi idual bu impo an oles [105].
E en in he deba e in ol ing COVID’s o igin, many scien is s seem o
imply ha he sec e o COVID in ec iousness and i ulence lies in he S
p o ein and ha he momen ha he sec e o S is unlocked, he o igin o
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COVID-19 will be unco e ed as well [102-103]. Con a y o he popula no ion,
he e is moun ing clinical and expe imen al e idence ha he S p o ein may
no be he main unde lying cause o COVID-19 in ec iousness and i ulence.
One majo piece o e idence o his lies in a comp ehensi e clinical s udy
conduc ed by Wol el e al [29]. In his s udy, i was ound ha COVID-19
pa ien s shed much mo e in ec ious pa icles han SARS-CoV-1 pa ien s. I we
look closely, he ques ion ha comes o mind is: why does SARS-CoV-2 need
much mo e pa icles o be mo e in ec ious i i s S has a 10 o 1000 imes [9-11]
g ea e binding a ni y o ACE-2 han he la e ? Keeping in mind ha i
equi es much mo e ene gy o p oduce hese ex a in ec ious pa icles and
na u e does no , as a ule, usually was e ene gy o do edundan hings. One
could, howe e , a emp o ge a ound his pa adox by claiming ha he S
a ni y o ACE-2 is esponsible o he mo e apid eplica ion o he i us.
Un o una ely, his sugges ion con adic s wha we know abou he li e-cycle
o he i us, as i al en y ep esen s only an ini ial s age o a long se ies o
p ocess ha includes RNA eplica ion, p o ein p oduc ion, assembly,
packaging, and he budding o i al pa icles [106-109].
The e a e hose who sugges ha i is possible ha SARS-CoV-1 S binds
mo e e cien ly o he ACE-2 in cells in he lowe espi a o y in con as o
SARS-CoV-2 [12]. The e a e a numbe o issues ha a ise when such as
a gumen is made. Fi s ly, he a gumen is made wi hou any conside ing
cu en physiological knowledge, namely he Mucocilia y Clea ance sys em
(MCC), which allows i al pa icles p oduced h oughou he espi a o y
sys em o be anspo ed upwa ds owa ds he nasal a ea so ha hey could be
expelled and shed [88-91]. Secondly, o ou knowledge, a emp s o ep oduce
he a gumen in ol ing SARS-CoV-1 S and ACE-2 has no been conduc ed
expe imen ally, p obably, because o he cu en di cul y in ob aining he now
ex inc SARS-CoV-1 o conduc compa a i e expe imen alongside SARS-CoV-
2. The e a e, howe e , also simila a gumen s made o accoun o he
di e ences in i ulence o Omic on and non-Omic on a ian s Hui e al [78].
This di e ence is ha COVID-19 pandemic p o ides us wi h a deluge o
clinical and expe imen al da a. In ac , a emp s o ep oduce his a gumen has
been made, which we will discuss a leng h la e .
The clinical s udy o Wol el e . al. [29] is jus he ip o he icebe g, as he e
is also a deluge o o he e idence ha shows ha he na u e o S is no wha
many scien is s ha e made i ou o be. No does S alone p o ide o a cohe en
concep ual amewo k o COVID-19 in ec iousness and i ulence, unlike M
and N. This is also he case wi h long COVID. We will examine hese in g ea e
de ails in la e sec ions. This is no o say ha S is unimpo an o ha i does
no play any pa in in ec iousness o i ulence. I is impo an , bu we mus
also ully comp ehend i s ue po en ial and limi a ion. A mo e comple e
unde s anding o he ue limi a ions and po en ials o S will come when we
s udy o he impo an p o eins such as N and M mo e ho oughly, which is
challenging e en i he majo i y o esea ch is o ien ed owa ds S.
4.2. SDMs and Rep oducibili y
We ha e seen ha SDMs a e highly ep oducible by hei abili y o
accu a ely p edic and explain ce ain phenomena ha a e o he wise di cul
o accoun o . Unlike al e na i e explana ions, hey a e explained in a cohe en
manne using a logical and uni ed pa adigm ha is consis en wi h cu en
knowledge o physiology and biochemis y [52]. The e is also impo an
clinical and expe imen al e idence ha ep oduces many o he p edic ions o
SDMs.
While a p e ious expe imen was no able o de ec any s a is ical
di e ence in he abili y o SARS-CoV-2 o las on a ious su aces unde
p esence o ligh when compa ed o SARS-CoV-1, Riddell e al. [21] conduc ed
a simila expe imen , de oid o ligh , and ound ha SARS-CoV-2 las s much
longe on ex e nal su aces han he con ol CoVs. SDMs p edic ha SARS-
CoV-2 is mo e pe sis en han mos i us as i s unusually ha d M p o ec s
agains he en i onmen and ha sh an imic obial enzymes, and his expe imen
ea ms SARS-CoV-2's esilience in he absence o ligh .
Rep oducibili y o SDMs is no con ned o compu a ional and
expe imen al esea ch, bu also ex ends o clinical s udies. One such s udy
in ol ed an in es iga ion in o he in ec iousness o SARS-CoV-19. I was ound
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ha COVID-19 pa ien s shed a much la ge amoun o in ec ious pa icles han
he 2003-SARS pa ien s [29]. This con adic s all o he pa adigms se o h. Fo
ins ance, i S is ully esponsible and has a much g ea e a ni y o human
ACE-2, why does SARS-CoV-2 need o expunge such highe quan i ies o
pa icles in o de o be mo e in ec ious? SDMs p o ide o a much mo e
elegan explana ion: he i us is mo e esis an o he sali a y and mucosal
an i-mic obial enzymes because o he abno mally ha d SARS-CoV-2 ou e
shell (low M PID). This clinical obse a ion aises mo e ques ions han i
answe s. Mo e speci cally, how is he i us able o make he hos expunge
such a la ge amoun o pa icles wi hou being mo e i ulen o he body? I we
assume ha he body is shedding mo e i al pa icles because i is p oducing
mo e i us copies, hen i al o gans such as he lungs should be ooded wi h
he i us, hus making he la e mo e dange ous, bu his is appa en ly no
happening in he case o COVID-19.
Adding o he pa adox, Ogando e al. [79] ound ha SARS-CoV-1 has a
highe i al g ow h VERO-E6 cells han SARS-CoV-2 unde he same
condi ions. E en i his is consis en wi h he g ea e pa hogenesis o SARS-
CoV-2, how do we hen econcile his esul wi h he p e iously men ioned
clinical obse a ion? I SARS-CoV-2 S has a 10 o 1000 imes [9-11] g ea e
a ni y o ACE-2 han he a ni y be ween SARS-CoV-1 S and ACE-2, how is
his possible? I seems ha we need o look elsewhe e o answe s by
examining N and M mo e closely. The SDMs explain ha he eason ha
SARS-CoV-1 is p oducing highe le els o pa icles has o do wi h he highe N
diso de ha allows g ea e e ciencies in i s eplica ion p ocess bu ,
con e sely, lesse in ec ious pa icles a e shed by he body as he highe M PID
does no p o ide su cien p o ec ion agains he an imic obial enzymes.
Ye ano he enigma in ol es long COVID [110-111], and SDMs a e able o
explain he pe sis ence o he i us among COVID-19 pa ien s e en mon hs
a e in ec ion. Again, we e u n o he heme o ha d M ha p o ec s he i us
om an imic obial enzymes. This ime he ocus is no only on mucosal and
sali a y enzymes as in he case o in ec i i y, bu also on an imic obial
enzymes in he immune sys em. Fu he discussion on he an imic obial
enzymes ound in he immune sys em can be ound in he long COVID sec ion
below.
4.3 Mo e Rep oducibili y: Omic ons and Pangolin-CoVs
The e a e many o he p edic ions ha SDMs make in ol ing N and M.
One such p edic ion pe ains o pangolin-CoV. When SDMs we e applied o
pangolin-CoVs, i became ob ious ha he 2017 pangolin-CoV (Pang2017)
isola e om Guangxi is a enua ed because o i s low N PID (~44%) [40,52,55-
56]. The e a e se e al implica ions o his nding. I his o a simila i us had
en e ed he human popula ion, i is likely ha i would had easily mo ed
quie ly among humans as a mild cold wi hou he no ice o he medical
communi ies [18]. The p edic ed a enua ion has been independen ly
ep oduced by se e al labo a o ies [52,55,74-77]. Animals models ha e seen
milde mani es a ion o symp oms upon Pang2017 in ec ion, in con as o he
Wuhan-Hu-1 s ain. The SDMs p edic ion pe aining o Pang2017 was
published be o e he a i al o Omic on.
Omic on was s de ec ed in Sou h A ica a ound No embe 2021.
Omic on was clinically and, la e , expe imen ally obse ed o be milde han
p e ious a ian s [71-78]. Once again, SDMs ha e much o say. Based on he
ini ial Omic on sub a ian , BA.1, he N and M PIDs a e 43.65% and 5.4%,
which a e bo h lowe han p e ious a ian s [49]. The smalle han usual M
PID (5.9% s. 5.4%) could sugges ha he i us had a ecen o igin in ol ing a
bu owing animal, whe eas he lowe N PID p edic s ha Omic on is likely
a enua ed o simila le els as Pang2017. The p edic ion in ol ing N PIDs was
expe imen ally eplica ed when i was shown ha he i al g ow h o VERO-
E6 cells in ec ed by Pang2017 and Omic on espec i ely a e e y simila , and
when i was shown ha he i al damage on cells by he wo i uses a e
simila [55] . This p esen s e idence o u he ep oducibili ies o bo h
Pang2017 and Omic on. Omic on has also been shown o be a enua ed wi h
lowe g ow h han Wuhan-Hu-1 unde i al i a ion [55,75,77,79].
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The co ela ions be ween N diso de and i al i e o i ulence can be
ound in SARS-CoV-2 ela ed i us da a ha include Pang2019 [71-73,75] and
he Lao ian ba s-CoV (BANAL) [50-51]. Fu he mo e, while Pang2017 has been
p edic ed and ea med o be a enua ed, his is no he case wi h Pang2019.
Ins ead, SDMs ha e o p edic Pang2019 o be non-a enua ed wi h i s N PID a
48.2% [18,52,55-56]. Huo e al. [81] we e able o ob ain le els o i al i e om
Pang2019 simila o hose o Wuhan-Hu-1 in VERO-E6 cells. Se e al
labo a o ies we e also able o independen ly obse e ha Pang2019 is able o
in ic se e e disease in a leas one s ain o mice. In con as , i ulence was
no obse ed in Pang2017 [55,74-79]. All hese a e again consis en wi h he
p edic ions o SDMs. We know ha all o hese SARS-CoV-2 ela ed i uses do
no possess FCS, unlike SARS-CoV-2. The ques ion hen becomes: how does
Pang2019 possess simila in ec i i y and i ulence as SARS-CoV-2 wi hou
FCS, when FCS-mu an non-Omic on SARS-CoV-2 is la gely no ansmissible
among e e s?
4.7. Measu ing Vi ulence Using CFR, Animal Models and Cell/Tissue Damage
Obse a ion
While Figu e 1 shows co ela ion be ween Inne Shell Diso de and CFR, i mus
be admi ed ha CFR is no necessa ily he mos ideal ep esen a ion o i ulence o a
leas wo easons. Fi s ly, CFR igu es a e o en ex apola ed ou o necessi y [103].
Secondly, CFR is applied only o human, no animals. Fu he mo e, i ulence may a y
among di e en animals e en wi hin a single i us o a ian . The e a e, howe e ,
o he me hods o measu ing i ulence such as animal models, i al i a ion and
indica ions o cell/ issue damage a e in ec ion. We ha e men ioned some o he
animal models. I mus be no ed ha a enua ion o agg essi eness o SARS-CoV-2
a ian s, Pang2017-CoV and Pang2019-CoV we e ep oduced by i al i a ions,
animal models and inspec ions o cell/ issue damage a e in ec ing cells o animals
we e conduc ed by a leas wo independen labo a o ies o each i us o a ian
[55,74-79]. The animal models included, a leas , hams e s and se e al s ains o mice.
In addi ion, he se e i y o Pang2019-CoV in ec ion was also obse ed in pangolins
unde labo a o y condi ions [80-82]. I can also be a gued ha a SARS-CoV-2 ela ed
i us may in ec di e en ypes o cells in di e en manne , which could po en ially
make in e p e a ion o i al i a ion da a mo e challenging. This is ela ed o he
a gumen made by some scien is s ha SARS-CoV-1 could in ec he lowe espi a o y
ac mo e e icien ly in compa ison o SARS-CoV-2. The e a e, howe e , hin s ha he
a gumen s may no p esen a g ea di icul y in he in e p e a ion o i al i a ion da a.
Vi al i a ions ha e been made a a ie y o cells including Ve o E6, Calu3 and Caco2
[77] . The da a show sub le bu de ini e di e ences in he i al i e s using he h ee
di e en cell lines ha is he esul o di e ences in S-ACE-2 binding mechanism. The
di e ences, howe e , a e no so huge ha i would make in e p e a ion o i al i a ion
da a challenging as he a ia ions i al i e s among cell ypes a e no g ea and ollows
a end. Besides, as we see la e , he S o SARS-CoV-2 ela ed i uses ypically
become quickly mo e e icien o binding o he S o di e en cell ypes a e se e al
passages.Wi h all hese in mind, i is possible o p esen a consis en pic u e o
i ulence nad in ec i i y. In ac , a p e ious pape [55] was able o ind a posi i e
co ela ion be ween i ulence and N PIDs based on a combina ion o da a om i al
i e , animal model and cell plaques.
5. Compa a i e Roles o S, M, and N P o eins
5.1. The S P o ein, Omic on, and Pangolins
We ha e seen ha he use o S is unable o explain some c ucial
expe imen al and clinical da a. This jus he ip o he icebe g, as he e is mo e
expe imen al and clinical da a ha S alone simply canno explain o accoun
o and, hus a , a emp s o do so a e no ep oducible, o can be shown o be
inhe en ly awed as we ha e a gued. The e a e also impo an biological
easons o his. Fo us o unde s and his, we need o examine he basic
undamen al biology o he i us. While S is de ni ely an impo an p o ein in
e ms o i al en y, he e is no undamen al eason ha S should be he mos
impo an p o ein in COVID-19 in ec iousness and i ulence, as much as many
scien is s would like o belie e. Vi al en y, hough impo an , is jus he s
o he many s eps in he i us' li e-cycle [109]. I mus be emphasized ha
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SARS-CoV-2 encodes 19 p o eins, e en hough no all a e majo p o eins [109].
S is no he only majo p o ein, and nei he is i he mos abundan p o ein. On
he con a y, he N and M a e he mos abundan majo s uc u al p o eins
ound in he cell and i ion espec i ely [96-100]. Fo his eason alone, we
should no be su p ised i N and M ha e g ea e in uence on he in ec i i y
and pa hogenesis o he i us.
Fu he se s o pe plexing e idence ha con adic he “S-alone”
pa adigm can be ound in he da a ha pe ains o pangolin-CoVs and
Omic on. We need o keep in mind ha all a ian s o SARS-CoV-2 ha e FCS
ha is absen in all COVID-19- ela ed ba and pangolin i uses. Many scien is s
hough ha hey ha e ound FCS o be he ue cause o COVID-19
in ec iousness and i ulence when i was ound ha SARS-CoV-2 wi h FCS
in ac is mo e agg essi e in espi a o y cells, in con as o he FCS-mu a ed
one, by p omo ing cell o cell usion. Fu he mo e, i has been shown ha while
non-Omic on SARS-CoV-2 wild- ype can be ansmi ed expe imen ally ia
ae osol be ween hams e s, no ansmission was seen when FCS-mu an was
used on e e s [113]. Again, how can his be he case, gi en ha pangolin-CoV
has no FCS and is easily ansmi ed be ween hams e s [74-75,81] ?
5.2. The S and Omic on Conund um
When Omic on a i ed, i p esen ed an enigma by being in ec ious and
ye a enua ed. How did Omic on achie e i s a enua ion gi en ha i has FCS
jus like all o he a ian s? Animal s udies ha e shown ha a s a e unable o
ansmi ia ae osol in he labo a o y. How could his be so when Omic on has
been clinically shown o be highly in ec ious? Mo e impo an ly, how can his
be happen when Omic on has e cien FCS [114-115], in which i s p esence in
o he a ian s has been shown o induce g ea e ansmissibili y in e e s
[113]? These disc epancies poin o he p obabili y ha o he ac o s a e in play.
In ac , all hese, as we ha e shown, a e consis en wi h he wo kings o N and
M, as summa ized in Figu e 5.
An a emp o ge a ound his “S-Omic on” pa adox is he hypo hesis ha
claims Omic on in ec s he cells in he uppe espi a o y sys em mo e easily
han he lungs. Hui e al. [78] we e able o isola e b onchial and lung issues,
which we e in ec ed wi h Omic on and p e ious COVID-19 a ian s. They
we e able o quali a i ely see he g ea e p esence o i al pa icles and
concluded ha Omic on is a enua ed because i eplica es mo e easily in he
lungs han in he b onchi. The e a e, howe e , a numbe o p oblems wi h his
in e p e a ion. Fi s ly, his obse a ion has no , o ou knowledge, been
ep oduced in o he labo a o ies. In ac , se e al labo a o ies ha e obse ed
e cien eplica ions in bo h lungs and uppe espi a o y sys ems [77,116-117].
Fu he mo e, se e al o he independen labo a o ies ha e no iced ha many
sub a ian s o Omic on a e e en mo e adap ed o he human ACE-2 han he
non-Omic on a ian s [116-118]. I his is he case, why hasn' Omic on
achie ed g ea e i ulence and in ec i i y like i s p edecesso s?
5.3. E idence o he Di e en Roles o N and M in Expe imen al Da a
The s o y is ac ually e en mo e complica ed han wha Hui e al. had
en isaged [78]. In eali y, he N and M p o eins, oge he wi h knowledge o
MCC, p o ide a mo e consis en and ep oducible explana ion o hei esul s.
In ou p e ious publica ions, we ha e shown ha he ini ial wa es o Omic on
ha lowe M PIDs han p e ious a ian s. This ea u e is likely a ell- ale sign o
i s ecen in e ac ions wi h a bu owing animal, possibly, he pangolin. In any
case, i explains why Hui e al. we e able o see mo e i al pa icles in he
b onchi. I is because Omic on is mo e esis an o he an i-mic obial mucosal
enzymes ha i encoun e s as i is being anspo ed upwa ds by he hai -like
s uc u es in he ne wo k o mucus-co e ed cilia y cells. The lung has no cilia y
cells o mucus bu has su ac an s ha , e en hough i is an i-mic obial, is no
as ha sh, unlike he a ie y o enzymes p esen . The appa en lack o i al
pa icles is likely an indica ion o pa icles s ill apped in he issues as he e
would no be MCC o b ing i o he op in he lungs [88-92] .
Vi al i a ions we e also made using Omic on, Del a, and Wuhan-Hu-1 in
lungs and b onchial issues in he abo e-men ioned expe imen . The da a we e
used o suppo he hypo hesis o di e en ia ed eplica ion o Omic on in he
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lungs and b onchi. In one o ou p e ious a icles, we managed o use hei
da a o pe o m mul i a ia e analysis (Figu e 6).The i al i a ion da a we e
aken om publicly a ailable pape o Hui e al, while N and M PIDs based on
a ian s and sub- a ian s we e aken om ou cu a ed diso de da abase
(Table 2). Reg ession (Mul i a ia e) Analysis was pe o med using R package.
The eg ession analysis p o ides us wi h he co ela ions be ween i al i e
and N/M PIDs is measu ed as co ela ion coe cien s ( ) o coe cien s o
de e mina ion ( 2). The o al n (sample size) o he en i e s a is ical expe imen
as seen in Figu e 6 is 24 (p < 00,1, n =24, 2~ 0.9). S a is ical esul s we e
compu ed using R package ha is a ailable publicly [119-120].
We ound s ong co ela ions be ween N/M PIDs and i al i e s. We we e also
able o obse e peculia changes in he signs o he co ela ions when mo ed
om lung issues o b onchial ones [40] . We we e able o ob ain a posi i e
co ela ion ( = + 0.96, Figu e 6A) especially on he N PID and i al i e (VT=
A * PIDN +B * Time + C ,whe e A,B,C = coe cien s and VT = i al i e ). I was,
howe e , e y s a ling when we ecei ed a nega i e co ela ion ( - -0,93,
Figu e 6B) be ween PIDM/PIDN and i al i e ( VT = A * PIDM+ B * PIDN + C *
Time + D whe e VT = i al i e , A,B,C = coe cien s and D = Y-in e cep ) [44].
Wha is ema kable and puzzling is he change be ween he posi i e and
nega i e signs. I is no only ep oducing he SDMs bu also ins uc ing us on
how o use he SDMs. The change in he sign is an indica ion ha he g ea e
p esence o pa icles in he lungs is dependen on he g ea e N diso de
(highe PIDN), whe eas he g ea e i al p esence in he b onchi is dependen
on lowe diso de in N and M PID (lowe M PID and N PID). The change in
he di ec ion o he slopes (co ela ions) can be obse ed in Figu e, 6C-D. We
will also see ha his change in slope (co ela ion) is comple ely absen when
i al i a ion is conduc ed in an animal model, ins ead o issues (Figu e. 7), as
i us pa icles can easily a el upwa d ia MCC in he case o animal models.
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Figu e 6. Mul i a ia e Analysis o M/N Diso de and Vi al Ti e . A. Reg ession analysis
e eals a posi i e co ela ion be ween N PID and he i al i e om human lung issues
and N PID. (Model: VT = A*(N PID) + B*Time + C whe e VT = Vi al Ti e , A,B =
Coe cien s , C = Y-In e cep .). B. Reg ession analysis ound a nega i e co ela ion
be ween i al Ti e om human b onchial issues and M/N PIDs. (Reg ession model:
VT = A*(M PID) + B (NPID) + C*Time + D whe e VT = Vi al Ti e , A,B.C = Coe cien s ,
D = Y-In e cep ). C. The h ee dimensional plane wi h he 95% con dence in e al as
applied o he eg ession analysis in ol ing i al i a ion on he lung issues. D. Th ee
dimensional plane wi h 95% con dence in e al ela ed o he he i al i a ion using
b onchial issues. As (C) and (D) p o ide only h ee dimensional ep esen a ions
(Model: VT = A*(N PID) + B*Time + C whe e VT = Vi al Ti e , A,B = Coe cien s , C =
Y-In e cep .), hey do no o e a comple e pic u e ha can only be ound in ou
dimensions(Reg ession model: VT = A*(M PID) + B (NPID) + C*Time + D whe e VT =
Vi al Ti e , A,B.C = Coe cien s , D = Y-In e cep ). The analysis is a s a is ical ex ension
o he expe imen o Hui e al. [40,43]. Da a pe aining o i al i a ion and PIDs a e
om he he expe imen o Hui e al and a ailable in Table 2 (which can also be ound
in p e ious publica ions) espec i ely. A nega i e slope (co ela ion) wi h espec o he
N PID-Ti e axes can be ound in (D), unlike (C).
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P io o his in es iga ion, we ocused on he ha d ou e shell (low M PID) in
esis ing he onslaugh o mucosal an imic obial enzymes and on g ea e N
diso de in i s abili y o assis in p o iding mo e e cien i al eplica ion [18-
20] , bu he expe imen also showed us ha N plays a ole in p o ec ing he
i ion om damage [52,55]. This is some hing ha we had o go en abou ,
e en hough we had e idence om he e y beginning ha ha de inne shells
do p o ec he i us om ha m in i uses such as EIAV, DENV, and abies [34-
35,55-56,65-67].
We conduc ed a simila eg ession s udy as seen in Figu e 7 using he
expe imen al da a o Guo e al. [75]. Again, we we e able o ob ain he i a ion
da a om he published pape o Guo e al wi h he espec i e N and M PIDs,,
which depends on he SARS-CoV-2 a ian and pangolin-CoV isola e, ob ained
om ou cu a ed da abase As wi h he da a om Hui e al, we did a eg ession
analysis o ob ain he co ela ion coe cien s ( ) and coe cien s o
de e mina ion ( 2) g ouped by he loca ion in he espi a o y sys em he
samples we e ob ained (Figu e 7, To al n = 30, p < 0.01, 2 ~ 0.9).
Guo e al. used Pang2017 and hams e s in lieu o Omic on and issue cul u es
espec i ely [65]. The e o e, M becomes an un eliable independen a iable as
he e is ha dly any di e ence be ween he M PID o Pang2017 and non-
Omic on SARS-CoV-2. We also expec ed o see he ull e ec o MCC since
Guo e al. [75] used an animal model, in con as o he use o issues by in Hui
e al. [78] i.e. he i us pa icles a e mo e able o mo e eely be ween di e en
pa s o he espi a o y sys em ia MCC. Posi i e co ela ions be ween N PID
and i al i e we e seen in samples collec ed om all h ee a eas o he
espi a o y sys em as seen in Figu e 7, which helps alida e SDMs and he
esul s desc ibed in he p e ious sec ions. Figu e 7B a ms a posi i e slope
(co ela ion) when iew wi h espec o he N PID-Ti e plane.
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oom o con o ma ion changes. The e o e, an M PID o 0 % means ha he i us is
no unc ional. The da a in Table 2 and Figu e 2 seem o ein o ce his idea by he
exis ence o he 4% minimum cu o poin . While a lowe M PID o e s g ea e
p o ec ion o he i us in i s na i e s a e, i could also imply ha M could ha e less
e iciency in unde going con o ma ional changes. We don' , howe e , know how much
impac his has on he e iciency o con o ma ional changes ha a e necessa y o
p ocesses such as i al en y and p o ein assembly. Ex apola ing om he e iciency o
SARS-CoV-2 (M PID ~ 6.3%) in eplica ion, i is likely no ha much, bu , again, his
is jus an ex apola ion ha has o be con i med by expe imen al esul s.
5.12. A Compa a i e Analysis o SARS-CoV-2, Pangolin-CoVs, and Lao ian Ba - CoV
Expe imen s Using S, N, and M
A mo e comp ehensi e analysis can be accomplished when examining M,
N, and S in expe imen al da a o SARS-CoV-2, ba -CoVs, and pangolin-CoVs.
We ha e seen ha all COVID-19- ela ed i uses a e po en ially in ec ious
because o hei abno mally ha d M. We ha e also seen ha Pang2019,
BANAL: (Lao ian ba -CoV) and Wuhan-Hu-1 a e all po en ially i ulen
because o hei high N PIDs (~48%), whe eas Pang2017 and Omic on a e
a enua ed as a esul o hei ela i ely low N PIDs (~44%). These ha e been
la gely ep oduced. Cells in ec ed wi h Pang2017 o Omic on ha e signs o
lowe i al g ow h and cy opa hic e ec s [55,74]. Simila ly, animal models
ha e shown less se e i y. In con as , howe e , i al i e s o cells in ec ed by
Pang2019 [80-82] and Wuhan-Hu-1 [75,79] a e highe han hose in ec ed by
Pang2017 o Omic on [55,74-76], jus as p edic ed by he SDMs. In con as ,
mice we e obse ed o be se e ely sickened by Pang2019 [81-82]. We need o
keep in mind ha his end is ue despi e he ac ha FCS is ound only in
SARS-CoV-2 including Omic on. The e o e, he s a k di e ences in he esul s
o he a ious expe imen s can only be accoun ed o when he oles o N and
M a e conside ed.
5.13. E idence o he Po en ials and Limi a ions o S: Vi al En y and Replica ion
The da a o he Lao ian ba -CoVs p esen an enigma [50-51]. I was
shown ha BANAL S binds o ACE2 in a di e en manne om SARS-CoV-2
e en as he BANAL i uses bind e cien ly o human cells. I onically, e en
hough he N PID o BANAL is close o hose o Wuhan-Hu-1 and Pang2019,
no se e e disease was de ec ed in mice in ec ed by BANAL-236. This seemed
inconsis en wi h wha SDMs p edic ed un il he da a we e s udied e y
ca e ully. I we inspec he i al i a ion da a, we can see ha he i al i e s in
VERO-E6 cells is high and compa able o Wuhan-Hu-1, bu his is no he case
in CALU-3 cells, whe e he di e ence is la ge ; i should also be kep in mind
ha a VERO-E6 cell is o kidney o igin, whe eas CALU cells a e espi a o y.
This implies ha BANAL-236 is mo e adap ed o kidney cells han espi a o y
ones p esumably because o i s S s uc u e. We also need o keep in mind ha
i has been obse ed ha he Lao ian ba -CoV S binds o ACE-2 in a di e en
manne [51].
While his se es as e idence ha S does play a ole in in ec i i y, i does
no show ha SDM esul s a e w ong o no ep oducible. Ins ead, i poin s o
he co ec way ha SDMs should be in e p e ed. Fi s ly, he expe imen al da a
emind us ha SDMs p edic po en ial i ulence, no necessa ily ac ual
i ulence, and his po en ial i ulence a ises om he high i al load in a leas
one o gan, which is popula ed wi h cells ha he i us can en e mo e easily. A
second lesson o be lea ned om he da a is ha SDMs p edic i ulence in
gene al, no jus humans, since N PID co ela es bes wi h highes i al i e s
among a ious cell ypes [55,74-79] , and high i al load is associa ed wi h
o gan ailu es. While human COVID-19 a ali y is mainly associa ed wi h
pulmona y (lung) ailu es, his may no be necessa ily so o o he animals.
The e o e, SDMs a e p edic ing po en ial i ulence in gene al, no jus in
humans.
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I he e is e idence ha S does play some ole in in ec i i y and i ulence
ia i al load, he ques ion hen becomes: how easily does S adap o bind
mo e e cien ly? I would seem ha g ea e S ness may be mo e easily
acqui ed han many belie e. Expe imen s ha e shown ha COVID-19 i uses
acqui e g ea e abili y o in ec he lungs a e passing o humanized mice
se e al imes [133-136]. Fu he mo e, a close compa ison o he wo may
p o ide clues. Gi en he ac ha bo h BANAL-236 and Pang2019 ha e high N
PIDs, why has Pang2019 been shown o be i ulen o humanized mice, unlike
BANAL-236? E iden ly, Pang2019 S is mo e adap ed han BANAL-236. To
unde s and how his is he case, we need o look a he e olu iona y
di e ences be ween he wo i uses. I we look a Figu e 5B, which is a
phylogene ic ee using M, we see ha Pang2019 is much mo e closely ela ed
o SARS-CoV-2 han BANAL-236. This implies ha Pang2019 was exposed o a
simila ange o hos s as Wuhan-Hu-1. Wha is also ema kable is ha bo h
BANAL-236 and Pangolin-CoVs, unlike SARS-CoV-2, do no ha e FCS. I is
likely ha Pang2019 spli o om SARS-CoV-2 o in ec mainly pangolins, and
as a esul los i s FCS while s ill main aining much o he es o i s S s uc u e.
This may also ha e implica ions o Peacock e al.’s FCS-mu an expe imen
[113] I is possible ha FCS-de cien S compensa es o i s de ci by binding o
ACE-2 in a di e en way o e he long un as in he case o Pang2019, and, as
we will see, i is easy o S o quickly adap o he ACE-2 o a pa icula species
o cell ype in a labo a o y.
I is e iden ha S has o be a leas su cien ly adap ed o ACE-2 in o de
o e en be in ec ious, bu in o de o SARS-CoV-2 o ha e any sus ained
in ec i i y o i ulence, o he ac o s mus also come in c ucial play, especially
he oles o M and N, as seen in he expe imen al and clinical e idence
pe aining o SARS-CoV-1/2. The ques ion hen becomes: how di cul i is o
S o gain su cien adap a ion o espi a o y cells? The ea lie sec ion a gues
ha i may no be ha di cul . Se e al s udies ha e shown ha COVID-19
i uses can easily acqui e adap abili y o human espi a o y cells in he
labo a o y e y quickly [133-136]. Acqui ing abno mally ha d M ha p o ides
o po en ially high in ec i i y, on he o he hand, may no be ha easy. I is
easy o nd CoVs ha e cien ly bind o S o espi a o y cells [101,109,118] o
ha e FCS [101,109,118] o ha S easily adap s [108,131-132], bu i is di cul o
nd CoVs wi h he abno mally ha d M, as mos CoVs a e no in ima ely
associa ed wi h a bu owing animal unlike COVID-19 ela ed i uses [4,16-
20,53-56] .
5.14. G ea e Model Rep oducibili y and Reliabili y Come When Mo e P o eins a e
Conside ed
The g ea e eliabili y and ep oducibili y o SDMs, in la ge pa , a ise
om he ac ha hey ake in o accoun he diso de o wo majo p o eins, M
and N, which a e mos abundan in he i ion and in ec ed cell espec i ely. In
ac , SDMs become un eliable i M o N is omi ed om conside a ion. SDMs
a e eliable and ep oducible in mos cases excep in ce ain occasions when
he ole o S has o be aken in o se ious conside a ion as we ha e seen in he
case o he Lao ian ba -CoVs. Fo his eason, we canno dismiss S o any o he
p o eins as unimpo an . In ac , as we ha e shown, SDMs become e en mo e
eliable and ep oducible when S (o o he p o ein) is aken in o conside a ion.
This end is consis en wi h wha we know abou he biology o i al
eplica ion. Vi al eplica ion in ol es mul iple p o eins e en i he e a e
p o eins ha play mo e majo oles han o he s.
5.15. Limi a ions and Po en ials o N, M and SDMs
We ha e al eady ouched on he limi a ion o SDMs. We ha e seen ha i
canno accoun some o he expe imen al esul s conduc ed using BANAL
(Lao ian ba -CoVs). They a e, o example, unable o accoun o he di e ences
in he he i al i a ions o BANAL-236 and SARS-CoV-2 on di e en cell
ypes (e.g. CALU, COCA, Ve o-E6) [51-52,77]. The signi cance o he
di e ences is, howe e , needs u he in es iga ion as o he esea che s ha e
shown ha SARS-CoV-2 becomes mo e e cien in eplica ion as se e al
passages a e made in each cell ype. Ne e heless, his poin s o he ole o S,
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which is a limi a ion o he SDMs. Cu en ly, SDMs in ol es on he i al shell
p o eins, which, in he case o CoVs, a e he N and M. Gi en ha SARS-CoV-2
has 29 i al p o eins and many o hem a e in ol ed in he eplica ion p ocess
[106-109], he e will be limi a ion i only one o wo p o eins a e used o s udy
in ec i i y, i ulence o long COVID. This is, o cou se, he case in SDMs,
which cu en ly use only M and N. In ac , as al eady men ioned, wi hou
ei he M o N, SDMs would be o limi ed ep oducibili y and eliabili y and
would ha e much di cul y explaining he unde lying cause o in ec i i y and
i ulence.
I onically, he ques ion is hen: How could M and N accoun o in ec i i y and
i ulence wi h e en a decen le el o ep oducibili y and eliabili y when he
limi a ion o using only wo p o eins is conside ed? The answe could lie in
wo ac o s. The impo an oles o M and N, as seen abo e, a e likely o be
pa ly esponsible. Secondly, a hin o an o e whelming impo ance o M and
N can be seen by he massi e abundance o he p o eins, no seen in o he
COVID-19 i al p o eins. This could also ein o ce he idea ha he wo
p o eins a e playing g ea e majo oles in he unc ioning o he i us. While
he ocus o his e iew pape is on SDMs, N and M, i is no in ended o
dismiss he impo ance o S o any o he i al p o ein. This e iew, howe e ,
a emp s o unde sco e he impo ance o M and N in in ec i i y and i ulence
as exempli ed by he exe cise seen in Figu es 6-7. The esul s seen in Figu es 6-
7 do no in alida e he ole o S, as Hui e al a emp ed o demons a e bu ,
a he , sugges ha he cu en ly unde s udied oles o M and N could e en
o e shadow ha o S wi h espec o in ec i i y, i ulence and, po en ially,
long COVID. O cou se, SDMs would become mo e ep oducible and eliable
i mo e i al p o eins such as S and NSP7 a e conside ed in he models, bu ,
cu en ly, SDMs ha e no each he s age whe e he oles o mo e p o eins can
be inco po a ed.
6. Pangolin Foo p in s and Long COVID
6.1. The Long COVID Enigma and Pangolin Foo p in s
One unusual cha ac e is ic ha is o en ound among some COVID-19
pa ien s is long COVID, which is when symp oms pe sis o weeks, mon hs, o
e en yea s a e he ini ial in ec ion {57-58,110-111]. While he cause o long
COVID emains la gely a mys e y, SDMs o e he mos logical and plausible
explana ion ye . We ha e seen ha all COVID-19 ela ed i uses ha e
ex ao dina ily ha d ou e shell (M) ha is no ound in any CoVs excep hose
associa ed wi h bu owing animals. We ha e also seen how SDMs explain ha
he ha d M allows o g ea e in ec iousness o COVID-19, by p o iding mo e
esis ance o mucosal and sali a y an imic obial enzymes, o en wi hou
g ea e i ulence ha is associa ed wi h g ea e N diso de [18-20,52,55-56].
Jus as a ha d M p o ides esis ance o an imic obial enzymes, i will almos
ce ainly also p o ide esis ance o o he des uc i e mechanisms o e ed by
o he aspec s o he hos immune sys em. This is possible as clinical s udies
ha e shown ha la ge amoun s o he i us end o emain in he body e en
a e mon hs [110-111]. We will u he explo e his link by a u he
examina ion o he a ious known aspec s o immunology ela ed o his
ma e as seen in he ollowing subsec ions.
6.2. Ha d M Resis ance o Vi olysis by Mac ophages and Complemen Sys em
In o de o u he examine he ole o an abno mally ha d M in long
COVID, we need o look mo e closely a how he immune sys em ge s id o
in ading o eign pa icles, especially i uses. Ou knowledge o immunology
helps us o ocus ou a en ion on p o eins p oduced by he complemen
sys em and lysosome [135-142]. These enzymes a e expe imen ally shown o
damage i uses and i al memb anes.
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Complemen p o eins a e p oduced mainly by hepa ocy es in he li e ,
e en hough hey a e sec e ed by monocy es, mac ophages, and epi helial cells
in he in es ines. The e a e, a leas , 30 ypes o complemen p o eins [139]. B
and T cells, along wi h an ibodies, can ale he complemen sys em in he
p esence o a o eign ma e such as bac e ia o i us. Complemen a y p o eins
assemble and bind o a p o ein a he a ge ed memb ane, and he p o ein
complex punches holes in he memb ane. Apop osis occu s in he case o
bac e ia o in ec ed cells, whe eas, in he case o a i us, his is e e ed o as
i olysis. I is a his poin in COVID-19 and long COVID ha he ha d M may
make i di cul o he complemen sys em o do i s job, as he p o eins a e
likely unable o pene a e he memb ane since M spans he en i e memb ane
ha co e s he i ion.
6.3. Resis ance o Vi olysis Wi hin a Mac ophage May P o ide he Vi us a Place o
Dwell: Possible Rese oi
A second way ha he immune sys em a emp s o ge id o pa hogens is
o expose hem o diges i e enzymes [130-131,140]. The mac ophage will s
engul he mic obe o mic obial p o ein, and upon phagocy osis, he immune
sys em will a emp o diges he pa icle ia lysosomes [138-141]. The p oblem
wi h his s a egy, howe e , is ha many pa hogens a e somehow esis an o
he exposu e o diges i e enzymes and end up li ing in he mac ophages
hemsel es [140-141]. An ongoing enigma pe aining o long COVID in ol es
he possibili y o a ese oi ha ha bo s he i us long a e he ini ial
in ec ion o he pa ien and whe e he sou ce ac ually is, since a ha d ou e
shell could p e en he mac ophage om des oying he i us and he i us
ends up li ing in he mac ophage. The obse a ion o an abno mally ha d M,
along wi h ou knowledge o immunology, sugges s ha he s place o look
a is none o he han he mac ophage i sel , and cu en esea ch suppo s his.
Huo e al. [144] ound he p esence o SARS-CoV-2 in he lungs o pa ien s
wi h symp oms o long COVID, whe eas se e al o he esea ch g oups ha e
ound p esence in o gans and issues h oughou he body [110-111], which is
consis en wi h he ac ha mac ophages can be ound in nea ly all o gans in
he body [139].
The na u e o SARS-CoV-2 should no be con used wi h ha o o he
i uses such as HIV and HSV. The ac ha SARS-CoV-2 has among he
ha des ou e shell, no jus among CoVs bu also among all i uses, is a ell-
ale sign ha he i us is o a di e en na u e om o he i uses such as HSV
and HIV-2 ha a e known o hide in places such as he b ain only o show up
la e [109]. HIV and HSV-2 ha e one o he mos diso de ed ou e shells
among i uses, unlike SARS-CoV-2. Thei highly diso de ed ou e shells help
hem pene a e and hide in o gans, as g ea e diso de allows o mo e e cien
p o ein-p o ein binding. SARS-CoV-2, on he o he hand, has one o he
ha des ou e shells. I i does no ha e he bene o a diso de ed ou e shell,
how does i hide? The answe has o lie in he mechanism o phagocy osis
desc ibed abo e. The e is esea ch showing ha in amma o y esponses may
be esponsible o long COVID. A ha d M and in lamma o y esponses such as
hose caused by cy okines a e no mu ually exclusi e [142-144]. Because o he
ex ao dina ily ha dness o M, i p o ides o a unique way o SARS-CoV-2 o hide in
mac ophages only o appea whene e he oppo uni y a ises. Whene e he i us
keeps eappea ing, he immune sys em could espond in such a way ha could esul
also in in lamma ion caused by cy okines.
6.4. G anuzymes: A Suspec ed Mechanism o M Resis ance
While he exposu e o des uc i e enzymes has been shown o ac agains
i uses in he complemen sys em and mac ophages, he e a e also o he
des uc i e enzymes a ailable in he immune sys em. These in ol e a amily o
enzymes known as g anuzymes. Upon en y o he i us, he immune sys em
will ini ia e a a ie y o de ensi e ac ions ha include Cy o oxic T-Cells and
Na u al Kille (NK) cells, which sec e e subs ances ha could po en ially
damage i al pa icles [136-139]. These cells sec e es g anuzymes in esponse o
a o eign in ade . One o he g anuzymes is pe o in, which binds o plasma
memb anes and punches holes ha allows o he g anuzymes o en e o cause
u he damage o he bac e ium o in ec ed cell [145]. While cu en
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expe imen al e idence has shown his o occu in memb anes o bac e ia and
in ec ed cells, he e is cu en ly no e idence ha i damages i al memb anes.
Ne e heless, gi en he known biochemical capabili ies o pe o in , i can be
cons ued ha pe o in can po en ially damage i ion in he same manne ,
since mos animal i uses including SARS-CoV-2 ha e a p o ec i e ou e
memb ane laye o p o ec hemsel es. In any case, pe o in causes lysis in
in ec ed cells, and he eby allows he i us pa icles o be exposed o he
complemen sys em and mac ophages. I is also known ha , while T-Cells and
NK cells sec e e pe o in, mac ophages sec e e pe o in-2 (PFN2), which is
simila o pe o in, bu i emains unclea i PFN2 a ec i al memb anes
di ec ly [144-145].
6.5. Uniqueness o COVID-19 S a egy o Immune E asion in Long COVID
The e a e many mys e ies in ol ing long COVID ha physicians and
scien is s a e s ill s uggling o sol e so ha we can come up be e ea men s.
How does SARS-CoV-2 induce long COVID? Wha a e he mechanisms? Is
he e a ese oi ? I so, whe e is i ? As we ha e seen, SDMs o e speci c
answe s o hese ques ions. While N diso de modula es he amoun o i us
eplica ed, especially in i al o gans, he unusually ha d M p o ides esis ance
o an imic obial enzymes. The e o e, he immune e asion s a egy used by
SARS-CoV-2 is d as ically di e en om ha o i uses such as HIV, HSV, and
HCV [4,16-20,65-67], which ha e high diso de in he o he shell ha allows
such i uses o hide in o gans. No only has no such high diso de been
de ec ed in SARS-CoV-2, he i us has one o he ha des ou e shell among
i uses, no jus CoVs. Tha is why he mac ophages o e he i us he
oppo uni y o dwell and hide in hem. Indeed, one labo a o y showed he
p esence o he i us in he lung al eola mac ophages o pa ien s who es ed
nega i e o he i us in he uppe espi a o y sys em [143]. Wha is e en mo e
puzzling is ha se e al o he labo a o ies ha e obse ed he p esence o he
i us in many o gans o long COVID pa ien s [111-112]. This aises he
ques ion, whe e is he ese oi ? Again, i mac ophages a e he ese oi , he
i us will be p esen in hese a ious o gans since mac ophages a e ound in
nea ly e e y o gan in he body.
6.6. Long COVID, long SARS and S
Long COVID has been linked o he ac i a ion o he immune sys em, ia,
pa icula ly, in e e ons (IFN-γ) and na u al kille T-cells (NK cells), ha
esul s in in amma ion [142,144]. Many scien is s poin o S as he main
unde lying cause o he ac i a ion. While his pos ula ion is e y in e es ing
and plausible, i aises mo e ques ions han i answe s. Fo ins ance, i doesn'
ell us he eason why some people ge long COVID, whe eas, o he s don' . I
doesn' ell us i long COVID a ises om a ese oi and, i so, wha is he
sou ce o he ese oi i.e. he hiding place o he i us. We also need o
emembe ha SARS-CoV-1 has a CoV S p o ein oo. Because he wo i uses
a e ela i ely closely ela ed (80%), i is likely he S o bo h i uses a e
unc ionally simila including hei abili y o ac i a e he immune sys em in
simila ways. A conund um is , howe e , seen when upon an examina ion o
he di e ences be ween long COVID and long SARS. One s a k di e ence is
he ac ha long SARS was usually associa ed wi h se e e mani es a ion o he
disease, whe eas long COVID could come e en wi h mild symp oms {64]. How
could he e be such a dispa i y i he S p o eins o he wo i uses a e likely o
be s uc u ally simila and when i has been shown ha S ac i a es he immune
sys em [142,144], which is likely o cause long COVID. SDMs o e s a no el
explana ion. SDMs ha e obse ed ha SARS-CoV-2 has a much ha de M han
ha o SARS-CoV-1 as a esul he i us is able o esis he immune enzymes
and hide in he mac ophage in he case o COVID. We a e, he e o e, likely o
ge a be e pic u e when we conside he oles o M. N and S keeping in mind
ha N also plays a ole as g ea e N diso de could allow g ea e p oduc ion o
i al pa icles e en in he e en o long COVID.
7. Summa y and Conclusion
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7.1. Rep oducibili y and Reliabili y o SDMs: Expe imen al and Clinical E idence
Some o he clinical and expe imen al e idence o he ep oducibili y and
eliabili y a e as ollows:
.i) The ex ao dina y ha diness o SARS-CoV-2 was seen expe imen ally by an
Aus alian g oup, Riddell e al [21]. They disco e ed ha SARS-CoV-2 las s much
longe in he en i onmen away om ligh han he CoV con ols.This is consis en
wi h he de ec ion o an ex ao dina ily ha d SARS-CoV-2 M by SDMs.
ii) The Du ch g oup, Ogando e al [79] [69] conduc ed a i al i a ion o SARS-CoV-2
ha was compa ed wi h SARS-CoV-1 and ound ha he i al i e s o SARS-CoV-1
a e a highe han hose o SARS-CoV-2. This is consis en wi h he SDM pe aining
o Vi ulence-Inne shell diso de , which p edic s ha SARS-CoV-1 will p oduce mo e
i al pa icles in cells and i al o gans and, he eby, making he i us mo e dange ous.
iii) The Ge man g oup Wol el e al [29] ound ha COVID-19 pa ien s shed
much mo e i al pa icles han SARS-CoV-1 pa ien s. Again SDMs show he
g ea es consis ency in explaining his. How do we explain he disc epancy
be ween (ii) and his disco e ed mechanism o in ec i i y? How can we
econcile g ea e in ec i i y wi h lesse i ulence and ice- e sa? The “S-
hypo hesis” says ha SARS-CoV-2 binds o ACE-2 wi h 10-1000 g ea e
a ni ies. I so, how do you accoun o (ii) and (iii)? SDMs explains ha e en
hough SARS-CoV-2 does no eplica e as e cien ly as SARS-CoV-1 as a esul
o i s lowe N diso de . Ii has a much ha de M ha is mo e esis an o he
sali a y and mucosal an i-mic obial enzymes and hus g ea e amoun o i al
sheddings occu .
i ) Why did Hui e al's a emp [78] o show ha Omic on (S) binds mo e
e cien ly o uppe espi a o y ac (ACE-2) ins ead shows high s a is ical
co ela ions wi h M and N PIDs jus as SDMs ha e p edic ed [86]?
) Why is Omic on much milde han p e ious a ian s bu ye as in ec ious as
o he a ian s? Once again, SDMs p o ide an elegan explana ion as Omic on
has lowe N PIDs and lowe o simila M PID when compa ed o o he
a ian s. The “S-hypo hesis” o e s an explana ion bu he e a e p oblems wi h
such an explana ion as seen in (i ).
i) Why was Pang2019 ound o be i ulen in animal models, i al i a ions
and cell plaques, whe eas he opposi e was ound in Pang2017? The S-
hypo hesis has no answe especially since all pangolin-CoVs lack FCS. SDMs
ha e al eady p edic ed hese e en be o e i s disco e y.
ii) Why do i al i e s o he a ious SARS-CoV-2 a ian s, SARS-CoV-2-
ela ed i uses and SARS-CoV-1 co ela e wi h hei N PIDs? Why do
a enua ion and i ulence o he a ious SARS-CoV-2 a ian s and SARS-CoV-
1 as de e mined by animal models and cell plaque s udies co ela e wi h N
PID?
iii) Wha is he na u e o long COVID? Again he S-hypo hesis has no answe ,
whe eas SDMs ha e.
ix) Why a e SDMs able o ie he di e en mani es a ions o COVID-19 ie
in ec i i y, i ulence and long COVID unde an umb ella o h ee closely
ela ed concep s? The S-hypo hesis, on he o he hand, s ill s uggles o
unde s and he ole o S in he h ee mani es a ions.
The e a e wo ac o s ha de e mine on how good a model is. These a e
ep oducibili y and eliabili y. Rep oducibili y e e s o he abili y o independen
labo a o ies ge ing simila esul s, whe eas eliabili y in ol es he abili y o
consis en ly explain and p edic he esul s [146-147]. The e o e, a model ha is unable
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o be eplica ed in di e en independen labo a o ies is un ep oducible, whe eas, a
model ha is unable o explain a esul is un eliable. (i)-(ix) summa izes bo h he
ep oducibili y and eliabili y o SDMs. We would a gue ha he e is de ini ely
e idence o bo h as we ha e seen e en hough, as we ha e seen, he e a e ce ain
occasions whe e o he p o eins such as S, ha e o be in ol ed in he explana ion.
While he expe imen al wo k o Riddell e al is c ucial in p o iding e idence ha
he abno mally ha d M o SARS-CoV-2 is likely o p o ide g ea e p o ec ion o he
i us, in con as o o he CoVs, he e is mo e ha could be done. Fo ins ance, he e
a e a a ie y o an i-mic obial enzymes ound in he mucus and sali a bu we know
only how hey in gene al can damage i uses . We s ill don' unde s and which speci ic
enzymes can damage o kill i uses SARS-CoV-2 o CoVs [22-28]. Expe imen s need
o be done on a ious CoVs wi h di e en le els M PIDs and a ious enzymes [22-28]
so ha we can ha e a be e unde s anding o he mechanisms in ol ed.
7.2. Links Be ween Vi ulence and In ec i i y
An impo an pa o ep oducibili y and eliabili y o a model o pa adigm is i s
abili y o explain ce ain esul s o ce ain phenomena. We ha e seen how SDMs a e
no only able o explain i ulence and in ec i i y bu also a e able o link in ec i i y and
i ulence. The wo mani es a ions a e ela ed as i ulence in ol es he abili y o he
i us o in ec cells in i al o gans such he i us is able o o e whelm i al o gans
especially he lungs, whe eas in ec i i y pe ains o i us' abili y o lea e he body o
in ec he cells o o he hos s. We can see ha he wo mani es a ions a e ela ed bu
how a e hey exac ly ela ed is complex. A conund um is quickly seen: Why is SARS-
CoV-2 less i ulen han SARS-CoV-1 bu is mo e in ec ious? How did he i uses
accomplish his? The S-alone hypo hesis is unable o p o ide a clea answe wi hou
u he complica ions and ques ions (see (i ) in 7.1 and MCC), whe eas he SDMs ha e
cohe en and clea cu answe s o his. Acco ding o SDMs. when he i us ha e highe
N diso de ( N PID), he i us is able o eplica e mo e quickly because g ea e diso de
a N p o ides o mo e e icien p o ein-p o ein/RNA/DNA/lipid binding and he e o e
mo e e icien and apid eplica ion. I ha is he case, hen why is SARS-CoV-1 less
in ec ious han SARS-CoV-2 since MCC en ail anspo a ion o much o he i al
pa icles o he nasal egion o be expelled ? Again, SDMs ha e a no el answe and i
has o do wi h he much ha de ou e shell (lowe M PID) ound in SARS-CoV-2 and
ela ed i us. This abno mally ha d M p o ec s he i al pa icles om he onslaugh o
an i-mic obial enzymes ound in he mucus and sali a e en i all SARS-CoV-2 a ian s
ha e lowe N PIDs i.e. less e iciencies in eplica ion o i al pa icles. This is
consis en wi h Wol el e al's clinical da a showing ha COVID-19 pa ien s shed much
la ge amoun o i us. As we can see, a mo e co ec app oach comes wi h a
coo dina ed unde s anding o he oles o M, N and S.
7.3. Pangolin Foo p in s in he E olu ion o COVID-19
The ph ase “pangolin oo p in s” e e s o a se o molecula signa u es
ha we e le behind by he in ima e e olu iona y in e ac ions COVID-19
ances al s ains had wi h pangolins. These signa u es ha in ol e he
abno mally ha d M and he end owa ds lowe N diso de a ose om he
pangolins' bu owing habi , which en ails a ha de ou e and o en inne i al
shell o acili a e o al- ecal ansmission ia bu ied eces. These ea u es o
pangolin oo p in s a e clinically mani es ed in he symp oms, in ec iousness,
and a enua ion/ i ulence o COVID-19. A e iew o cu en knowledge o
immunology indica es ha i is also mani es ed as long COVID. The
abno mally ha d M ha acili a es g ea e in ec iousness by being esis an o
an imic obial enzymes in he sali a and mucus could also esis a emp s by
he immune sys em o ge id o i in o he ways, hus leading o long COVID.
7.4. Clues Poin ing o Pangolin Foo p in s in he E olu ion o COVID-19
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A unique and highly unusual p ope y ha in ol es all examined SARS-CoV-2-
ela ed i uses is i s ha d ou e shell (low M PID). This p ope y is e y a ely ound
e en among he CoV amily. Wha is he e olu iona y o igin o his peculia
cha ac e is ic? A e ospec i e sea ch o ou diso de da abase o CoVs p o ided us
wi h a clue when i was ound ha e y ew CoVs ha e such excep ionally ha d M and
hose ound we e associa ed wi h a bu owing animal such as abbi s. Fu he mo e, i
can be seen ha phylogene ic ees using M ha e a much close ela ionship o SARS-
CoV-2 han p e ious phylogene ic ees ha e shown. How can his be e en possible
when RaTG13 has 96.4% o SARS-CoV-2, whe eas pangolin-CoVs ha e
app oxima ely only 90% simila i y o SARS-CoV-2? Wha is e en mo e puzzling is
ha one o ou ees shows ha Omic on has a close ela ionship o pangolin-CoVs
han o he o he a ian s. The answe has o do wi h he ac ha phylogene ic
algo i hms do no handle ecombina ions well and M may be he bes choice o
phylogene ic s udies since M is abno mally s uc u ed (low diso de ), which means ha
i is likely o be highly conse ed. Secondly, many scien is s ha e been looking o an
in e media y animal hos o SARS-CoV-2 o no a ail. I has ob iously no occu ed o
many scien is s ha he e is no in e media y animal hos is likely because he ances al
i us has en e ed and e-en e ed he human popula ion and o he animal popula ions
o e a long ime o ime wi h a p ima y o seconda y ese oi being pangolins. This
could explain how all SARS-CoV-2 ela ed i us ha e unusually ha d M and SARS-
CoV-2 is highly in ec ious no jus o humans bu also o a wide ange o animals. I
akes ime o he i us o become highly adap ed o such a wide a ie y o animals. All
hese p esen clues o a mo e unique e olu iona y ela ionship be ween pangolins and
SARS-CoV-2 ha needs o be u he esea ch as he e a e hin s ha he esul ing
cha ac e is ics a e ela ed o he beha io s and clinical mani es a ions o he i us.
7.5. G ea e Rep oducibili y and Reliabili y When M, N and S a e Used in
Coo dina ion
The e is hus a no cu en e iew a icle based on upda ed da a
a emp ing o demons a e he ep oducibili y ha S is he main p o ein o sole
p o ein esponsible o he in ec i i y o pa hogenesis, especially when
compa ed o o he i al p o eins such as M and N. As we ha e shown, he e is
indispu ably impo an clinical and expe imen al e idence ha shows ha S
canno accoun o he di e ence in i ulence and in ec i i y be ween SARS-
CoV-1 and SARS-CoV-2, e en hough he e is de ni ely some e idence o i s
ole in modula ing in ec i i y including an ibody e asion ia S mu a ion. The
beha io s o N and M ia SDMs can accoun o much o he clinical and
expe imen al esul s and a e highly ep oducible e en o he smalles de ails.
The e a e impo an undamen biological easons o his. While S is
impo an o i al en y and an ibody ecogni ion [109], i is no he mos
abundan p o ein. N and M a e he mos abundan p o eins in he in ec ed cell
and i ion, espec i ely [109].
Fu he mo e, i al en y is only he ini ial, albei highly impo an , s ep in
he mul is ep p ocess o he i us li e-cycle. We ha e, howe e , also seen ha
he oles o S in in ec i i y and i ulence become clea e and mo e consis en
when we examine o he p o eins, especially M and N, alongside S. The
in e playing oles among he a ious p o eins ha e o be conside ed be o e we
can ully unde s and he ac ual na u e o hese p o eins and hei impac s on
in ec i i y and pa hogenesis. Wi hou S’s g ea e adap ion o ACE-2, he e
would be ine cien i al en y, i any, bu wi hou he ha d M and a ying N
diso de he e would no be any highly sus ainable in ec i i y o he di e se
le els o i ulence espec i ely seen in COVID-19.
Also, as men ioned, S alone canno accoun o he di e ences in
in ec i i y and i ulence be ween SARS-CoV-1 and SARS-CoV-2 wi hou
u he ques ions, gi en cu en clinical and expe imen al da a. N and M, on
he o he hand, do p o ide a cohe en ly no el explana ion ha should be
u he explo ed. I is also wo h no ing ha many labo a o ies ha e shown
ha i easy o S o adap o he ACE-2 e en wi hin espi a o y cells [133-135],
bu i is ex emely di cul o nd CoVs wi h such a ha d M as in he case o
SARS-CoV-2- ela ed i uses [4,18-20,52-54]. Acqui ing such ha d M which is
necessa y o po en ial high in ec i i y had o ake i s ime o e ol e om he
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in e ac ions wi h a bu owing animal, i.e. pangolins, and none o he high
COVID in ec i i y we ha e seen would ha e been possible wi h his c ucial
e olu ion. Fu he mo e, i has been a gued ha no hing is unique abou SARS-
CoV-2, including i s FCS, since i is no di cul o nd CoVs wi h simila
p ope ies. In ou da ase , we a e, howe e , unable o nd CoVs wi h such an
abno mally ha d M among CoVs no associa ed wi h a bu owing animal, and
he e a e e y ew CoVs associa ed wi h a bu owing animal, i we don' coun
COVID-19- ela ed i uses.
7.6. Long COVID
Fu he mo e, S is no able o e a plausible hypo hesis o he cause o
long COVID. The cause o long COVID has hus a emained a comple e
mys e y, which is a g ea hind ance o he sea ch o mo e e ec i e
ea men s. The M and, o a much smalle ex en , N ia SDMS, howe e , o e
an elabo a e and highly plausible explana ion using ou cu en knowledge o
immunology on he cause o long COVID: he ha d ou e shell o he i us is
likely o make i di cul o mac ophages, T-Cells, and o he such en i ies o
ge id o he i us. As a esul , he e may be plen y o oppo uni ies o he
i us o dwell in he mac ophages, which a e a likely ese oi .
7.7 Clues o Fu he Resea ch
We ha e seen ha SDMs o e s a no el cohe en ha links i ulence and
in ec i i y wi h N and M ia p o ein in insic diso de . Bo h expe imen al and
compu a ional e idence o he eliabili y and ep oducibili y [146-147] o SDMs
as applied o COVID-19 is co e ed. We ha e ied o show ha SDMs using N
and M a e by and la ge ep oducible when expe imen al and clinical da a
especially om o he labo a o ies a e sc u inized. The eason o
ep oducibili y and eliabili y o M and N can a guably be aced o he
abundance o he majo p o eins and hei impo an oles in he eplica ion
p ocess. The ole o M in in ec i i y is based on he obse a ion o an
abno mally ha d SARS-CoV-2 M using AI. In e es ingly, phylogene ic s udy o
SARS-CoV-2 ela ed i uses poin s o a close ela ionship be ween pangolin-
CoVs and SARS-CoV-2. This has no been shown in any o he phylogene ic
s udy using o he p o eins o en i e genome. While e idence o he
ep oducibili y and eliabili y o SDMs as applied o COVID-19 using N and M
has been p esen ed, u he expe imen al and clinical esea ch is needed o e-
a m hei ep oducibili y and eliabili y [146-147].
E en hough M and N a e he mos abundan p o eins in he i ion and
cell espec i ely, he e a e 29 CoV i al p o eins, o which many a e also
in ol ed in eplica ion and i ulence. Fo his eason, we ha e ied o show
ha much o he limi a ions o COVID-19 SDMs a ise om his ac . A solu ion
would be o include mo e p o eins such as S and NSP7 as discussed. This is
whe e u he esea ch is also necessa y since he cu en SDMs inco po a e
only N and M.
While we ied o a gue using he exis ing amewo k o SDMs and
cu en knowledge o immunology ha SDMs as applied o long COVID is
p omising , he models as cu en ly applied o long COVID a e s ill
p elimina y o a i s in ancy and, he e o e, ely on ci cums an ial e idence.
Gi en ha long COVID is s ill la gely a mys e y, i is impe a i e ha long
COVID be u he esea ched in o using no el means including SDMs.
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`
Au ho Con ibu ions: Concep ualiza ion, G.K.-M.G.; da a cu a ion, G.K.-M.G. and
V.N.U.; o mal analysis, G.K.-M.G.; in es iga ion, G.K.-M.G.; me hodology, A.K.D. and
G.K.-M.G.; esou ces, A.K.D. and J.A.F.; so wa e, G.K.-M.G.; alida ion, G.K.-M.G.;
isualiza ion, A.K.D. and J.A.F.; w i ing—o iginal d a , G.K.-M.G. ; w i ing— e iew
and edi ing, V.N.U.
Funding: This esea ch ecei ed no ex e nal unding.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable..
Da a A ailabili y S a emen : In his sec ion, please p o ide de ails ega ding whe e
da a suppo ing epo ed esul s can be ound, including links o publicly a chi ed
da ase s analyzed o gene a ed du ing he s udy. Please e e o sugges ed Da a
A ailabili y S a emen s in sec ion “MDPI Resea ch Da a Policies” a
h ps://www.mdpi.com/e hics. I he s udy did no epo any da a, you migh add
“No applicable” he e.
Con ic s o In e es : G.K.M.G. is a esea ch scien is and he owne o Goh’s
BioCompu ing, Singapo e. He has w i en a book, “The Vi al Shapeshi e s: S ange
Beha io s o HIV and O he Vi uses” [4]. The au ho s decla e no o he con ic o
in e es .
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