2019 Recent Aspects on the Pathogenesis Mechanism, Animal Models and Novel Therapeutic Interventions for Middle East Res

2019·3.06 MB·English

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REVIEW
published:26March2019
doi:10.3389/fmicb.2019.00569
Recent Aspects on the Pathogenesis
Mechanism, Animal Models and
Novel Therapeutic Interventions for
Middle East Respiratory Syndrome
Coronavirus Infections
SinoshSkariyachan*,SnehaBasavarajChallapilli,SwathiPackirisamy,
SupreethaToplarKumargowdaandVaishnaviSnehaSridhar
R&DCentre,DepartmentofBiotechnology,DayanandaSagarCollegeofEngineering,Bengaluru,India
Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an emerging zoonotic
virus considered as one of the major public threat with a total number of 2 298
laboratory-confirmed cases and 811 associated deaths reported by World Health
Organization as of January 2019. The transmission of the virus was expected to
Editedby:
be from the camels found in Middle Eastern countries via the animal and human
KelvinTo,
UniversityofHongKong,HongKong interaction. The genome structure provided information about the pathogenicity and
Reviewedby: associated virulent factors present in the virus. Recent studies suggested that there
ManLungYeung,
were limited insight available on the development of novel therapeutic strategies to
UniversityofHongKong,HongKong
MahmoudMohamedShehata, induce immunity against the virus. The severities of MERS-CoV infection highlight
NationalResearchCentre,Egypt the necessity of effective approaches for the development of various therapeutic
*Correspondence: remedies. Thus, the present review comprehensively and critically illustrates the recent
SinoshSkariyachan
aspects on the epidemiology of the virus, the structural and functional features of
[email protected];
[email protected] the viral genome, viral entry and transmission, major mechanisms of pathogenesis
and associated virulent factors, current animal models, detection methods and novel
Specialtysection:
strategies for the development of vaccines against MERS-CoV. The review further
Thisarticlewassubmittedto
Virology, illustrates the molecular and computational virtual screening platforms which provide
asectionofthejournal
insights for the identification of putative drug targets and novel lead molecules toward
FrontiersinMicrobiology
thedevelopmentoftherapeuticremedies.
Received:28October2018
Accepted:05March2019
Keywords:MERS-CoV,emergingzoonoticvirus,mechanismsofpathogenesis,animalmodels,probabledrug
Published:26March2019
targets,vaccinedevelopment
Citation:
SkariyachanS,ChallapilliSB,
PackirisamyS,KumargowdaSTand INTRODUCTION
SridharVS(2019)RecentAspectson
thePathogenesisMechanism,Animal The coronaviruses such as HCoV-229E, HCoV-NL63 (α-coronavirus) and HCoV-OC43 and
ModelsandNovelTherapeutic HKU1(β-coronavirus)circulatedinthehumanpopulationandcausedmildrespiratoryinfections
InterventionsforMiddleEast
(HamreandProcknow,1966;Fouchieretal.,2004;vanderHoeketal.,2004;Wooetal.,2005).
RespiratorySyndromeCoronavirus
Infections.Front.Microbiol.10:569. Over the last two decades, there have been two zoonotic emergence of coronaviruses into the
doi:10.3389/fmicb.2019.00569 humanpopulation,bothlinkedwithSevereAcuteRespiratorySyndromeCoronavirus(SARS-CoV)
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(Drosten et al., 2003; Kuiken et al., 2003) and Middle East This outbreak provides an exclusive opportunity to seal the
Respiratory Syndrome Coronavirus (MERS-CoV) (Zaki et al., gap in our awareness of MERS-CoV infection (Oh et al.,
2012;ChafekarandFielding,2018). 2018). Recent statistics suggested that as of 31 December
MiddleEastRespiratorySyndromeCoronavirusbelongedto 2018, the total of 2,279 laboratory-confirmed cases of MERS-
β-coronavirus,clade-c(vanBoheemenetal.,2012;deGrootetal., CoV with 806 associated deaths (case fatality rate: 35.4%)
2013)andwasinitiallyknownasHumanCoronavirusErasmus were reported in 27 countries [Harcourt et al., 2018; World
Medical Center/2012. or HCoV-EMC/2012 (Chan et al., 2012). Health Organization (WHO), 2018a]. Among various cases,
MERS-CoV infections resulted high mortality rate in human, 1901 confirmed cases with 732 related deaths (fatality rate
and till date, very limited therapeutic intervention or vaccine 38.5%) werereported from SaudiArabia [Harcourtet al., 2018;
are available for the treatment of infections caused by the virus WorldHealthOrganization(WHO),2018a].
(Mustafaetal.,2017;Huietal.,2018). In a comprehensive report by WHO, 1841 laboratory cases
Middle East Respiratory Syndrome Coronavirus found in were confirmed between 2012 and 2016 in which 80% of them
various natural hosts such as the dromedary calves (Camelus were reported from Kingdom of Saudi Arabia. The remaining
dromedarius), bats (Vespertilio superans and Neoromicia cases were reported from 27 countries in Middle East, Asia,
capensis),andEuropeanhedgehog(Erinaceuseuropaeus)(Zhang North Africa, United States of America, and Europe [World
et al., 2016; Lau et al., 2017). The replication of the virus has Health Organization (WHO), 2016a]. A total of 13 cases were
observed in various other animals such as rabbit, goat, civet, reported between October to November 2017, amongst which
pig, camelid, and horse (Muller et al., 2012; Chan et al., 2013a; 12 cases were reported in male and only one case was reported
Al-Tawfiqetal.,2014;Eckerleetal.,2014;Meyeretal.,2015).The in female. Most of the infected individuals were hospitalized
prevalence MERS-CoV was recently reported in non-camelid duetothecontactwithcamelsandconsumptionofcamelmilk
domestic mammals such as sheep, goat, donkey, and cow with the virus [World Health Organization (WHO), 2017a].
that were in contact with camels (Kandeil et al., 2019). The Additionally,twolaboratorycaseswerealsoreportedinwhichthe
origin of human MERS-CoV is considered to be from the bats. patientsbelongedtothecountriessuchasMalaysiaandUnited
Several coronaviruses were isolated and found that which were Arab Emirates [World Health Organization (WHO), 2018a].
genetically similar to human MERS-CoV. Phylogenetic analysis The laboratory confirmed cases and deaths reported in several
of MERS-CoV showed that a close relationship to Pipistrellus countriesfromtheyear2012to2018areshowninTable1.
batcoronavirusHKU5andTylonycterisbatcoronavirusHKU4
(Selingeretal.,2014).Studieshavesuggestedthattherewerefew
evidences where the virus showed camel origin (Zhang et al., THE GENOME OF MERS-CoV
2016). Studies revealed that the sero-prevalence of the virus
was greater among the people those who were exhibited direct Intheearlyyearsof2000,coronaviruseswerenotacquiredgreat
andindirectcontactwithdromedarycamelswhencomparedto importanceinresearchanddevelopment.Withtheemergenceof
those who were exhibited with the general population (Watson SARSintheyear2002andMERSin2012,theinterestofmany
et al., 2014; Zhou et al., 2015; Conzade et al., 2018). Recent researcherswasstimulatedtowardcoronavirus(Perlman,2015).
study revealed that the prevalence of MERS-CoV infection The morphology of coronavirus includes spherical or
in camel workers was found to be very high in Saudi Arabia pleomorphic shapes with a diameter of 80–120 nm (Masters,
(Alshukairi et al., 2018). Thus, this review provide the recent 2006).Thegenomeofthecoronavirusconsistsof6and7open
perspectives on the epidemiology and genome composition of reading frames (ORFs). The ORF 1a and 1b encompass two-
MERS-CoV with a special emphasis on the viral entry, clinical third of the viral genome which encodes the non-structural
manifestations, mechanism of pathogenesis, animal models, poly-proteins and the other four ORFs on the downstream
detection, and development of vaccines with an insight on side encode for the structural proteins such as envelope
variouscomputationalbiologyperspectives. protein (E), Spike protein (S), nucleocapsid protein (N), and
membraneprotein(M).Insomecoronaviruses,hemagglutinin-
esterase (HE) gene is present in the region between ORF 1b
EPIDEMIOLOGY and S (Marra et al., 2003; Rota et al., 2003; Snijder et al.,
2003). These structural proteins are folded and entered in to
The first case of MERS-CoV infection was reported in the endoplasmic reticulum (ER) and transported to the ER-
Saudi Arabia in 2012 (Zaki et al., 2012). From the period of Golgi transitional slot (Masters, 2006). During the replication
its emergence, the virus has infected and propagated across of coronavirus, substantial amounts of structural proteins are
more than 20 countries, contributing to a wide number of synthesized in order to assemble the progeny virions (Fung
laboratory-confirmed cases. In 2015, the MERS-CoV outbreak and Liu, 2014). They occupy the RNA genome which encodes
in South Korea involved 186 cases which included 38 fatalities. structural proteins such as S protein, M protein, and N protein
A total of 83% cases of transmission were due to five super (Snijderetal.,2003).
spreaders,and44%of186caseswereinthepatientsthosewho The genome size of the first imported MERS-CoV strain
were experienced nosocomial transmission at 16 hospitals. The was identified to be 30,114 nucleotide (nt) long, including
epidemic lasted for 2 months and the government authorities the 3(cid:48)and 5(cid:48) UTRs. This strain showed the genome structural
quarantined16,993personsfor14daystomanagetheoutbreak. ornaisationof typicalbetacoronavirus suchas a5(cid:48)-untranslated
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TABLE1|LaboratoryconfirmedcasesofMERS-CoVinfectionsanddeathsreportedinseveralcountriesfromtheyear2012to2018.
Year Region MERS-CoVinfections DeathsduetoMERS-CoV Reference
infections
2012 SaudiArabia,Qatar,andJordan 9 3 WorldHealthOrganization,2012
2013 SaudiArabia,UnitedKingdom,France,Tunisia, 167 71 WorldHealthOrganization(WHO),2013a
andItaly
2014 SaudiArabia,UnitedArabEmirates,Jordan, 765 273 WorldHealthOrganization,2014
Iran,andEgypt
2015 SaudiArabia,RepublicofKorea,Jordan, 680 237 WorldHealthOrganization(WHO),2015a,b
Kuwait,ThePhilippines,Thailand,UnitedArab
Emirates,Oman,Qatar,China,andIran
2016 SaudiArabia,UnitedArabEmirates,Qatar, 243 75 WorldHealthOrganization(WHO),2016b
Lebanon,andOman
2017 SaudiArabia,UnitedArabEmirates,Qatar, 258 81 WorldHealthOrganization(WHO),2017b
Lebanon,andOman
2018 SaudiArabiaandMalaysia,UnitedArab 96 41 WorldHealthOrganization(WHO),2018a
Emirates
region (UTR) (nt 1 to 272), replicase complex ORF1ab (nt An interesting fact of coronaviruses is that they display
273 to 21508), S gene (nt 21450 to 25511), ORF3 (nt 25526 recombination, high rates of mutation, and propensity to cross
to 25837), ORF4a (nt 25846 to 26175), ORF4b (nt 26087 species. One such event was reported during the 2002–2003
to 26827), ORF5 (nt26834 to 27508), E gene (nt 27584 to epidemic, where the transmission of SARS-CoV has occurred
27832), M gene (nt 27847 to 28506), N gene (nt 28560 to from Chinese horseshoe bats to human populations (Perlman
29801),ORF8bgene(nt28756to29094),and3(cid:48)UTR(nt29094 and Netland, 2009). MERS-CoV has the capability to adjust
to 30114). The two main poly-proteins such as pp1ab and new environments and acquire various virulence factors and
pp1a are cleaved into 15/16 non-structural proteins (nsp) by enhance their ability to transfer these factors from human-
3C-like protease (3CLpro) and papain-like protease (PLpro). to-human due to continues outbreak (Zumla et al., 2015).
These proteases are cleaved from pp1ab along with other ORFs The phylogenetic analysis revealed that all human and camel
encodingnspwhichareessentialforactivatingRNAdependent MERS-CoVs were identified to be homologous to each other.
RNApolymerase,helicase,exo-ribonuclease,endo-ribonuclease, Additionally, the hedgehog and bat MERS-CoVs demonstrated
and methyltransferase. These nsp were identified to be nsp12, para-phyletic group to human and camel MERS-CoV clade
nsp13, nsp14, nsp15, and nsp16. The nsp14 is necessary for (Zhangetal.,2016).
proofreading the ubiquitous mutation of RNA virus (Ziebuhr Kim (2016) reported that the occurrence of a mutant strain
et al., 2000; Snijder et al., 2003; Gorbalenya et al., 2006; Smith of MERS-CoV toward the human CD26 receptor during the
et al., 2013; Raj et al., 2014; Durai et al., 2015). Studies SouthKoreanoutbreak.Furthermore,theyisolated13newviral
revealedthattheaccessoryORFproteinsplayanimportantrole genomesinwhich,12possessedpointmutationinthereceptor-
in MERS-CoV infection and pathogenesis (Menachery et al., bindingdomain(RBD)ofSprotein(Kim,2016).TheSprotein
2017). This study showed that the absence of major accessory is suggested to be essential for the host tropism through its
ORFs such as deletion of ORF3, -4a, -4b, and -5 (dORF3-5) interaction with the host CD26 receptor (van Doremalen et al.,
played major role in the viral replication and pathogenesis. 2014;Wangetal.,2014).
Further, the attenuation of the mutant dORF3-5 was found
to be responsible for the dysregulated host responses such as
PATHOGENESIS
augmented interferon (IFN) pathway activation, disrupted cell
processes, and robust inflammation. Thus, the disruption of
accessoryORFsprobablyprovideplatformtotheattenuationof Viral Entry
future emergent strains of MERS-CoV accessory ORF mutants. The entry of MERS-CoV to the host is facilitated by its type-
TheaccessoryORFfunctionscanbetargetedforboththerapeutic I transmembrane glycoprotein known as S protein (Xia et al.,
and vaccine treatments in response to MERS-CoV and related 2014). The RBD of MERS-CoV consists of S1 subunit which
group2Ccoronaviruses(Menacheryetal.,2017).Thegraphical rangedfrom367to606aminoacidsandcanbegroupedintoan
representation of the genome organization of MERS-CoV is externalsubdomainandcoredomainasshowninFigures1C,D.
illustrated in Figure 1. Coronaviruses have demonstrated the The receptor-binding motif, V484 – L567 of RBD is situated at
establishment of double membrane vesicles (DMVs) in the the external sub domain and the core sub domain consists of
infected cells. Based on immune-histochemistry and electron anti-parallelβ-sheet(five-stranded)andsixhelicesinthecenter.
microscopic studies, the coronavirus replicase proteins are co- Thetwosmallβ-strandsmakeaglobularfold.Thecoredomain
localized with the DMVs and are assumed to be the site of structure is balanced by three disulfide linkages. The MERS-
replication/transcriptioncomplex(FungandLiu,2014). CoV RBD external sub domain contains a β-sheet along with
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FIGURE1|(A)GraphicalrepresentationofthestructureoftheMERS-CoV.TheschematicrepresentationshowsthatthestructureofMiddleEastRespiratory
SyndromeCoronavirus,whichcontainstheRNAenclosedinthenucleocapsidwithintheenvelopethatisembeddedwithspikeproteins,glycoproteinsand
membraneproteins(vandenBrandetal.,2015).(B)GenomeorganizationofMERSCoV.ThegenomicorganizationofMERS-CoVdisplaystheviralgenesS,3,4a,
4b,5,E,M,8b,andN.Thegenomeencodesforfiveuniqueaccessoryproteinssuchas3,4a,4b,5and8bandfourmajorstructuralproteinswhichareillustratedin
thegenomescheme(Gaoetal.,2016).(C)ExperimentalstructureofMERS-CoVcomplexeswithhumanDPP4(PDBID:4L72).DPP4isanextracellularRBD
domain,whichcomprisesofbothN-andC-terminal.TheN-terminalistheβ-propellerdomainandC-terminalistheα/βhydrolasedomain(Wangetal.,2013).(D)
Genomeorganizationofreceptorbindingdomain(RBD)thatillustratestheS1andS2sub-domains(Zhangetal.,2015).
one small and three large strands which are organized in anti- reported that various antiviral peptides interfere the formation
parallel manner. The RBD core is attached to this region by 6HB(Fornietal.,2015).
specialinterveningloopsanditbindstothecoresub-domainby
aclampatthelowerandupperregions.Mostoftheconnecting Transmission and Clinical Presentation
loops and two small 310 helices are located in the inner region TheclinicalpresentationofMERS-CoVcompassesfromsevere
of the sheet. The fourth disulfide linkage is stabilized among respiratory diseases to subclinical infections (Drosten et al.,
the residues starting from C503 to C526 and β6-strand linked 2013; Hui et al., 2018). Infected patients often indicate the
withη3-helix.Threehelices(HR1)atthecenterandthreechains presence of hemoptysis, sore throat, fever, cough, shortness
(HR2) near to the core promote the release of the viral particle of breath, and other gastrointestinal symptoms such as
intothecytoplasmandpromotetheprogressionoftheinfection diarrhea and vomiting (Albarrak et al., 2012; Assiri et al.,
(Duraietal.,2015). 2013a,b; Guery et al., 2013; Health Protection Agency, 2013;
MiddleEastRespiratorySyndromeCoronaviruscanalsoenter Omrani et al., 2013). A low range of lower pulmonary
the cell through an auxiliary pathway on the cell surface via infiltrate associated with viral pneumonia was observed in
transmembraneproteases.ThebindingofMERS-CoVintohost the radiograph of patients infected with MERS-CoV (Memish
cell and replication of the virus is shown in Figure 2. The et al., 2013a; Wang, 2014). It was observed that greater than
host protease cleaves coronavirus S protein into two functional 60% of the initially reported cases of MERS-CoV infection,
domains distinctive to each other at the N-terminal region the patients experienced severe disease which demanded
(denoted as the S1 subunit) which comprised of RBD, and intensive care treatments like extracorporeal membrane
C-terminal portion (S2 subunit) comprised of a fusion peptide, oxygenation and mechanical ventilation. Hematological
two heptad repeats (HR2 and HR1) and the transmembrane aberrations delineated for the clinical cases of neutrophilia
(TM)domain(WeissandNavas-Martin,2005).Themembrane (8%), lymphopenia (34%), thrombocytopenia (36%), and
fusion is conciliated by main conformational rearrangement lymphocytosis (11%). While the conditions of kidney failure
which exposed the fusion peptide resulting in the development necessitaterenalreplacementtherapyforaconsiderablenumber
ofsix-helixbundle(6HB).Thecoreof6HBconsistsofatriple- of MERS-CoV cases, several studies detected MERS-CoV
stranded coiled like structure constructed by HR1s of the three antigens and particles in the renal tissues in vivo, which
spike subunits forming trimers; the HR2 elements are found to is the direct indication of the virus replication in renal
bepackedwithinthegroovesofthecoiledcoilinananti-parallel tissues and long term acute infection (Yeung et al., 2016;
direction.Owingtotheircentralroleinmembranefusion,itwas Alsaadetal.,2018).
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FIGURE2|LifecycleofMERS-CoVdisplayingfusionwithplasmamembrane.ThefusionofSproteintotheplasmamembraneofhostcell,formationofadouble
membranevesicleinthehostcell,eventuallyreleasingtheRNAenclosedinthenucleocapsidfollowedbygenometranscription.TheviralRNAundergoesreplication
andtranscriptionfollowedbythe4,5,and6RNAsynthesisandtranslation;theendoplasmicreticulumaidstheassemblyandpackagingofvirusparticleforminga
completedoublemembranevesicleandlastlythroughexocytosisandMERS-CoVisreleasedoutofthehostcell(Duetal.,2009).
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The first evidence of human-to-human transmission of The serological analysis and extensive investigation of those
MERS-CoV was reported in few cases in United Kingdom, potentiallyexposedtothepatientssuggestedthattherewereno
when an adult male traveled to Saudi Arabia and transferred secondaryinfections[WorldHealthOrganization,2013b].Asa
the infectious virus to two of his family members (Perlman minimum of 18 cases of asymptomatic MERS-CoV infections
andNetland,2009).ItwassuggestedthatMERS-CoVinfections were reported in health care personals, the function of these
were due to the introduction of the virus in humans and subclinical cases in the transfer of infection was found to be
the close contact with camels was one of risk factors for the uncertain(deSousaetal.,2014).
transmissionofMERSasreportedbyWorldHealthOrganization
[WorldHealthOrganization(WHO),2016b].
Molecular Mechanism of the
ThetransmissionofMERS-CoVisdeterminedasspasmodic,
often healthcare associated, intra-familial, and required Pathogenesis
prolonged and close contact (Memish et al., 2013a,b, 2014a,b; Whiletherewereintensivestudiesoncoronavirusresearchover
Puzelli et al., 2013; Drosten et al., 2014). In a domestic study thepast5years,limitedreportsareavailableonthepathogenesis
conducted with MERS-CoV infected patients, 14 out of 280 of MERS-CoV. Recent post-mortem histopathological studies
contacts of 26 positively indexed patients (antibody positive or revealedthatthelocalizationofviralparticlesinthepulmonary
RNA) suggested that the frequency of the transmission even and extra pulmonary tissue of a 33-year-old male patient of T
during outbreaks was approximately 3% (Drosten et al., 2014). cell lymphoma, who acquired MERS-CoV infection (Ng et al.,
Several studies suggested that the virus was not self-sustained, 2016; Alsaad et al., 2018). The histopathological analysis of the
as during local epidemic of MERS-CoV, which has not been biopsieswerecollectedfromlung,brain,liver,heart,kidney,and
readily transmitted to more than one human and has been skeletalmuscledemonstratedthattherewerepulmonarydiffuse
reportedinmajorityofhumanrelatedcases(BauchandOraby, alveolar damage, necrotizing pneumonia, acute kidney injury,
2013; Breban et al., 2013; Cauchemez et al., 2014; Chowell portal and lobular hepatitis and myositis with muscle atrophic
and Nishiura, 2014). The mode of transmission of the virus is changes. Further, the viral particles were found to be localized
illustratedinFigure3. in the pulmonary macrophages, pneumocytes, renal proximal
FIGURE3|TransmissionofMERS-CoVandsymptomspossessedbyinfectedhuman.Thefigureshowsthepathbywhichthevirusistransmittedfromtheinfected
cameltothehumanandcameltovariousanimals(Kandeiletal.,2019);further,thisvirusistransmittedtootherhumanpopulationviahumantohuman
transmissions(Shehataetal.,2016).
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tubular epithelial cells and macrophages infiltrating the skeletal crystal structure of DPP4 is illustrated in Figure 1C. A DPP4
muscles(Alsaadetal.,2018). bindingprotein,adenosinedeaminase,isconsideredasoneofthe
Studies suggested that the pathogenesis of MERS-CoV in majorinhibitorsoftheSproteinofMERS-CoV(Drosten,2013).
human and animals are mainly due to three mechanisms such A number of cell lines such as human-derived HFL, Calu-3,
as DPP4 (dipeptidyl peptidase-4) mediated mechanism, papain Caco-2, Huh-7, HEK, His-1 cell lines, CD8+, and CD4+ that
likeproteasePLpromediatedmechanismandaccessoryproteins aresusceptibletoMERS-CoVwerereported(Chanetal.,2013b;
likep4aandmembraneMproteinmediatedmechanism.Hence, Shiratoetal.,2013;Chuetal.,2016).TwoSproteinecto-domains
these proteins are probably considered as one of the potential can be divided into fusion-catalyzing domains (FD) and RBD.
therapeutic targets for MERS-CoV infections (Durai et al., The S RBDs bind to DPP4 after which the FDs are exposed
2015).Themolecularmechanismsexhibitedbyvariousmodesof through unfolding. Further, the cellular and viral membranes
pathogenesisareillustratedinFigure4. werejoinedtogetherbyrefolding(Rajetal.,2013).Theunfolding
FDrequiredthecleavageofSproteinbyhostproteases.Outof
various proteolytic cleavage sites between the FD and RBD, the
DPP4 MEDIATED MECHANISM cleavageatS1/S2siteisessentialfortheinfectionofCalu3cells
byMERS-CoV(Parketal.,2016).
DPP4 is a type-II transmembrane glycoprotein consists of It has been revealed that upon infection, monocyte chemo
approximately 766 amino acids and function as major receptor attractant protein-1 and IFN-γ-inducible protein-10 were
for MERS-CoV. The three dimensional structural analysis induced and suppressed the proliferation of human myeloid
revealed that this receptor has α/β-hydrolase domain and a progenitor cells (Zhou et al., 2014). MERS-CoV can infect
β-propellerdomainwitheightbladesthathelpedinthebinding T-cellsfromhumanlymphoidorgansandcausestheperipheral
ofreceptorbindingdomaininMERS-CoV(Rajetal.,2014).The blood inducing apoptosis by intrinsic and extrinsic pathways
FIGURE4|ThemolecularmechanismofpathogenesisbyMERS-CoVinhumanandanimals.ThepathogenesismechanismismainlyoccurredbyDPP4,papian
likeproteasePLpro,andaccessoryproteinssuchasp4aandmembraneMprotein(Duraietal.,2015).
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(Chuetal.,2016). A study conducted by Al-Qahtani et al. thenucleus.Intheabsenceof4b,expressionofpro-inflammatory
(2017) suggested that the inhibition of DPP4 mitigated the cytokines was observed as NF-κB translocated to the nucleus.
induction of PPARγ (the transcriptional repressor) and IRAK- This was also observed in the case of 4b mutants that devoid
M(negativeregulatorofTLRsignaling)whichindicatedthatthe ofnuclearlocalizationsignal,indicatingthatnuclearlocalization
immunosuppressiveactionofSglycoproteinregulatedbyDPP4. signal mediated process of 4b is essential for NF-κB expression
(Cantonetal.,2018).
Asacomponentoftheviralenvelope,Mproteinisinvolved
PAPAIN LIKE PROTEASE PLpro
in various functions such as viral assembly along with other
MEDIATED MECHANISM proteinssuchasE,S,N.TheM,E,andSproteinsinteractwith
NproteinintheER-Golgicomplex.Thisinteractionhindersthe
Upon entry into the cells, two ORFs at the 5(cid:48)-end of the fusion of viral and cellular membranes (Mustafa et al., 2018).
viral genome are translated into two poly-proteins. They It was observed that the expression of M protein in MERS-
are processed by two viral proteases, PLpro and 3C-like CoV might suppressed the type-I IFN expression in response
proteinase (3CLpro) and form 16 nonstructural proteins topoly(I:C)inductionorSendaivirusinfection.Thisreaction
which are necessary for the membrane-associated replication was detected to be specific for the activation of IFN regulatory
complex. PLpro is located in nonstructural protein 3. PLpro factor 3 (IRF3), however, not activated the nuclear factor-κB.
was found to be multifunctional enzymes with deISGylating The interaction of MERS-CoV M protein with TRAF3 and the
(removal of ISG15 conjugates from host cell factors) and de- associationofdisruptedTRAF3–TBK1leadtoreducedactivation
ubiquitinating (cleavage of ubiquitin from host cell factors) ofIRF3(Luietal.,2016).
propertiesinadditiontoproteaseactivityinmanycoronaviruses
(Clasman et al., 2017). This resulted in the host antiviral
immune response being antagonized and promoting the viral HOST IMMUNE RESPONSE
replication. PLpro can inhibit the mitochondrial anti-viral
signaling protein induced IFN-β reporter activity and reduce Middle East Respiratory Syndrome Coronavirus can readily
TNF-α induced NF-κB reporter activity. Hence, MERS-CoV infect human respiratory epithelial cells, macrophages, T-cells,
PLpro is considered to be important antiviral target (Mielech dendriticcellsandcaninfluencetheproductionandinductionof
etal.,2014;Harriganetal.,2018). pro-inflammatorycytokinesandchemokines(Zhouetal.,2015).
The coronavirus genome is encoded by two ubiquitin-like Studies on ex vivo human lung tissue and respiratory epithelial
domains (Ubl1 and Ubl2), out of which Ubl2 is considered to cell lines showed that the induction of antiviral interferons
beanimportantcomponentwhichinfluencesthePLproactivity. followed by MERS-CoV infection by stimulated epithelial cells
Clasman et al. (2017) reported that PLpro has not showed was found to be limited. A study by Menachery et al. (2014)
the deISGylating, de-ubiquitinating or protease activities in the describedthatthecellsinfectedwithMERS-CoVdemonstrated
absenceofUbl2domain.Therewerenovariationsintheprocess distinctlyalteredchromatinstructuressuchasrepressivehistone
ofinhibition,substratespecificity,catalyticefficiencyandthermal markers,thatcouldlimitthetranscriptionfactorsfrombinding
stability(Clasmanetal.,2017). to the promoter regions of interferon-stimulated gene. The
changes in DNA methylation found to be one of the reasons
fordownregulationofIFN-γ–associatedantigen-presentinggene
ACCESSORY PROTEINS MEDIATED
aftertheinfection(Menacheryetal.,2018).MERS-CoVproteins
MECHANISM such as 4a, 4b, M, and PLpro were found to be suppressed the
induction of interferons (Yang et al., 2013; Mielech et al., 2014;
The viral genome is recognized by melanoma differentiation- Cantonetal.,2018).Further,theinductionofpro-inflammatory
associated protein-5 (MDA5), retinoic acid inducible gene-1 cytokineresponseinthesecellswasfoundtobelimited.Adelayed
(RIG-1)andendosomaltoll-likereceptor3(TLR3)aspathogen- inductionofpro-inflammatorycytokines/chemokinessuchasIL-
associated molecular patterns. This recognition resulted in the 6, IL-8, and IL-1β were observed upon the viral infection (Lau
formationoftype-1interferon(IFN1).Asanevasionmechanism, etal.,2013;Cantonetal.,2018).
virussynthesizeproteinsthathindertheproductionIFN1inthe MiddleEastRespiratorySyndromeCoronaviruscouldinfect
pathway(ZinzulaandTramontano,2013). and replicate in human monocyte-derived macrophages and
In the absence of MDA5 and RIG-1, MERS-CoV used the immature monocyte-derived dendritic cells, while it failed
protein such as 4a (p4a) which contain RNA-binding motif for to perform similar functions in mature monocyte-derived
binding (Rabouw et al., 2016). By a direct binding, this motif dendriticcells.Thisindicatethatthematurationrestricttheviral
masksthedsRNAofMERS-CoV.Thep4apossessesαβββαfold replication. Immature dendritic cells were unable to stimulate
withtheβ1-β2loopandα1helixwhichbindtotheminorgroove T-cell even after the antigen uptake, delaying the activation
of dsRNA. The mutational studies revealed that p4a-dsRNA of T-cells and permitting further viral replication (Cong
complex stabilized by the amino acids such as K27, W45, K63, et al., 2018). The infection of human macrophages induced
and K67 (Batool et al., 2017). In a study carried out by Canton the expression of pro-inflammatory cytokines/chemokines
etal.(2018)suggestedthat,NF-κBremainedinthecytoplasmof such as MCP-1/CCL-2, IFN-α2, IFN-γ, MIP-1α/CCL-3, IP-
MERS-CoVinfectedcellswhile4bwasfoundtobeattachedwith 10/CXCL-10,RANTES/CCL-5,IL-8,TNF-α,IL-12p40,andIL-6
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(Zhouetal.,2015;Congetal.,2018).Theinfecteddendriticcells musthaveananalogousfunctioninthediseasemodelasinthe
inducedtheexpressionofIL-12,RANTES/CCL-5,IP-10/CXCL- clinicalcondition(Denayeretal.,2014).
10andIFN-γ,however,showedlowexpressionofIFN-αandno Anidealanimalmodelistheonewhereanimmunocompetent
expressionofIFN-β(Zhouetal.,2015). animal, on receiving the challenge virus at a realistic dose
T-cells can be directed to the infection site by type-I through a suitable inoculation route, is able to replicate
IFN stimulated secretion of IL-10 and CXCL10. Due to their the characteristics of a human disease as closely as possible
uncontrolled and strong expression, the expression of IFN- (Doremalen and Munster, 2015). Ferrets, hamsters, mice and
γ and IL-12 were inhibited and restricted the activation of other small laboratory animals are not ideal animal models for
T-helpercells.Withtheaddedeffectofdown-regulatedantigen- MERS-CoVowingtothevariationsinDPP4receptor.Therhesus
presentation pathways, the activation of T cells is inhibited. macaque and the common marmoset are the first non-human
Hence,thesequesteredTcellsfailedtotargetthevirus(Yingetal., primate species (NPS) to be used as the animal models for
2016).Previousstudiesrevealedthatwhentheserumsamplesand MERS-CoV.GlycosylationofmouseDPP4(mDPP4)preventsits
bronchoalveolar lavage were analyzed from two patients, it was binding to S protein of MERS-CoV. Hence, transgenic hDPP4
observedthatthepatientswithlowlevelsecretionofIFN-αwere (human DPP4) mice were developed as animal model (Peck
failedtosurviveandthepatientsdisplayedhighlevelsecretionof etal.,2015).ThemajoradvantagesandlimitationsofMERS-CoV
type-IIFNweresurvived(Faureetal.,2014). animalmodelsarelistedinTable2.
ANIMAL MODELS Mice
Studies suggested that the glycosylation of certain amino acids
The animal models are one of the vital components for prevented the binding of mouse DPP4 (mDPP4) to S protein
translation of the findings from drug discovery process from of MERS-CoV (Doremalen and Munster, 2015). The first
benchtobedside.Theseanimalmodelsareindispensableforthe transgenicMERS-CoVmousemodelwasestablishedbyAgrawal
deeper understanding of any diseases and safe and protective et al. (2015). The mice expressed hDPP4 and developed severe
vaccine development process (Lorenzen et al., 2015). The main respiratoryMERS-CoVinfections.Thetransgenicmiceinfected
criteriaforthevalidationofanimalmodelsinpreclinicalstudies withanintra-nasaldoseof106TCID50ofMERS-CoVdeveloped
arefacevalidity,predictivevalidityandtargetvalidity.Theface pneumonia,weightloss,acutepulmonaryviralinfection,ruffled
validity means that the proper similarity between the animal fur,squinting,anddeathwithinfewdays(Agrawaletal.,2015).
model and the human disease in terms of the biology and A transgenic mouse model developed by Zhao et al. (2015)
symptoms of the disease. However, assessing face validity is a showed that limited utility for pathogenic studies due to the
tedioustaskduetothelackofunderstandingonthecorebiology systemicdisseminationanddevelopmentofsevereneurological
ofthediseasesymptoms.Predictivevaliditymeansthedisplayof diseases.Theotherlunginfectionmodelswerelackmortalityand
clinicalinterventionswhichexhibitedsimilareffectintheanimal weight loss, however, developed lung pathology in response to
models.Thisisalsoverydifficulttoachieveduetotheincomplete 50%TCID50ofMERS-CoV(Taoetal.,2015).
correlation between animal models, the mechanism of human Recentstudiessuggestedthatthetransgenicmicemodelswere
diseasesandtheincapabilityofstandarddrugstoactiveinmany developedwherethefull-lengthmDPP4genereplacedbyhDPP4
animalmodels.Targetvalidityisanotheressentialcomponentfor (Coleman et al., 2017). The infection and replication of MERS-
thevalidationofanimalmodelsinwhichthetargetunderstudy CoV was observed in the lungs, however, disseminated slow
TABLE2|AdvantagesandlimitationsofMERS-CoVexperimentalanimalmodels.
Species Advantages Limitations Reference
Rabbit Readilyavailableandeasytohandle Animal-to-animaltransmissionstudiesarenot Haagmansetal.,2015;
availableNoclinicaldisease,Lowviraltitersin Vergara-Alertetal.,2017
tissuesuponinfectionDevelopmildpulmonary
lesions
Rhesusmacaques Human-specificreagentsavailableforimmunologic LimitedavailabilityandexpensiveExpert Baseleretal.,2016;Yuetal.,
analysisImmuneandrespiratorysystemssimilarto husbandryrequirementsAnimal-to-animal 2017
humans;clinicaldiseasesimilartohumansUseful transmissionstudiesarenotavailableTransient
forconfirmingvaccineefficacytesting diseaseEthicalconcerns
Commonmarmoset Modelsevere,potentiallyfatalMERS-CoVinfection LimitedavailabilityandexpensiveExpert Vergara-Alertetal.,2017;Yu
Somehuman-specificimmunologicalreagents husbandryrequirementsEthicalconcerns etal.,2017
cross-reactRespiratoryandimmunesystems Animal-to-animaltransmissionstudiesarenot
similartohumans;clinicaldiseasesimilarto available
humansUsefulforconfirmingantiviralandvaccine
efficacytesting
hDPP4-transgenicmice Modelsevere,potentiallyfatalMERS-CoVinfection GlobaloverexpressionofhDPP4 Baseleretal.,2016;Coleman
EasytohandleReagentsavailableUsefulfor etal.,2017
screeningantiviralsandvaccines
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impact to the other organs during the post injection with dose infectedrabbitsdemonstratedthepresenceofMERS-CoV.They
range of 102–105 PFU. The pathology of infected mice showed generally develop respiratory diseases such as asymptomatic
pneumonia-likesymptoms(Colemanetal.,2017). andmildhumaninfectionsassometimesobservedinimmune-
The CRISPR-Cas9 gene editing tools were used to produce competentpatients(Haagmansetal.,2015).
mice susceptible to MERS-CoV infection by modifying Recently, Houser et al. (2017) revealed that the infected
their genome sequence (positions 288 and 330) homologous New Zealand rabbits were developed asymptomatic pulmonary
to the sequence of hDPP4. In certain mice model the infections with high levels of viral antigens and RNA. Multiple
characteristic symptoms such as pulmonary hemorrhage, lung lobes showed peri-vascular inflammation. The antibodies
low survival, decreased pulmonary function and weight loss developed by the rabbits lacked neutralizing activity, and
were observed. The 288-330+/+MERS-CoV mouse model as a result the animals were susceptible to re-infection. In
demonstratedsevererespiratoryinfectionasobservedinhumans fact, enhanced pulmonary inflammation was observed after
(Cockrelletal.,2016). re-infection. However, re-infection elicited the neutralizing
antibodies(Nabs).Hence,itwassuggestedthatuponre-infection,
Rhesus Macaques and Marmosets there might be greater risk of severe lung disease in those
who do not develop neutralizing antibody response, or those
Upon infection with MERS-CoV, rhesus macaques and
whoseneutralizingantibodytitershavedecreasedaftertheinitial
marmosets were developed moderate respiratory disease
infection(Houseretal.,2017).Theclinicalsignsandlesionsalong
which are comparable with mild human MERS-CoV infection.
withtheoccurrenceofMERS-CoVRNAandantigensobserved
As observed in human cases, they also showed complete
inMERS-CoVinfectedanimalmodelsareshowninTable3.
blood count abnormalities. Rhesus macaque and marmoset
models were found to be suitable for pathogenic studies of
mild infections. Both the models can be used for the efficacy
DIAGNOSTIC APPROACHES
testing of prophylactic and therapeutic countermeasures
(Baseleretal.,2016).
The primary diagnosis of MERS-CoV infection was performed
Yuetal.(2017)reportedthatwhen2-3oldrhesusmacaques
by molecular techniques such as real-time reverse transcriptase
and common marmosets were intra-tracheally injected with
PCR (RT-PCR) (Corman et al., 2012a), reverses transcription–
hCoV-EMC,hematoxylinandeosinstainedtissuesoftheinfected
loop-mediated isothermal amplification (RT-RTPA) (Shirato
modeldemonstratedlesionsprimarilyinthelungswithvarious
etal.,2014)andreversestranscription-recombinasepolymerase
degrees of inflammation, hemorrhaging, pulmonary oedema,
amplification (RT-LAMP) (Abd et al., 2013). Numerous
interstitial pneumonia, eosinophil infiltration and necrosis in
serological assays were used to detect MERS-CoV or closely
bronchial epithelial cells and pneumocytes. The lungs of
related viruses in seropositive camels; these tests were protein
the infected marmosets demonstrated widespread pulmonary
microarrays like indirect enzyme-linked immunosorbent assay
oedema and hemorrhaging. The oedematous alveolar cavities
(ELISA), recombinant spike immunofluorescent assay, spike
showed fibrinous exudates and neutrophil infiltration. In both
pseudoparticle neutralization and microneutralization assay
the cases, DPP4 was widely expressed in alveolar macrophages
(Buchholzetal.,2013;Pereraetal.,2013).However,noneofthe
and type-I and II pneumocytes were also actively involved.
serological assays showed evidence of the precise occurrence
The pathological changes as a result of viral infection were
of MERS-CoV in camels (Song et al., 2015). Recent study
not observed in any other organs of the infected animal
reported that a rapid and specific assay for the detection of
(Yuetal.,2017).
MERS-CoV such as nucleic acid visualization technique which
combine the reverse transcription loop-mediated isothermal
Rabbits
amplification technique and a vertical flow visualization strip
In most cases, MERS-CoV causes severe infection in the lower (RT-LAMP-VF) to detect the N gene of MERS-CoV (Huang
respiratory tract in humans. Nasal swabs collected from the et al., 2018). The study suggested that in comparison with the
TABLE3|ClinicalsignsandlesionsalongwiththeoccurrenceofMERS-CoVRNAandantigensasseeninMERS-CoVinfectedanimalmodels.
Animalmodel Clinicalsigns Lesions Occurrenceof Occurrenceof Reference
MERS-CovRNA MERS-antigen
Rabbit Asymptotic Rhinitiswithnecrosis Lung,upperrespiratory TypeIandII Haagmansetal.,2015
tract,lymphnodes pneumocytes
RhesusMacaques Mildtomoderate Interstitialpneumonia Lung,lymphnodes, TypeIandII deWitetal.,2013a,b;
respiratorydisease upperrespiratorytract pneumocytes,alveolar Yaoetal.,2014;Yu
macrophages etal.,2017
CommonMarmoset Mildtosevere Broncho-interstitial Lung,blood,lymph TypeIpneumocytes, Falzaranoetal.,2014;
respiratorydisease pneumonia nodes,visceralorgans, alveolarmacrophages Yuetal.,2017
upperrespiratorytract
hDPP4-transgenicmice Severefatalrespiratory Broncho-interstitial Lung,brain, TypeIandtypeII Agrawaletal.,2015
disease pneumonia visceralorgans pneumocytes
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