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8/3/2019 Benh Chan Tay Mieng

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EnterovirusesPan, Tingting

Laboratory of Medical Molecular

Virology, Fudan University


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Enterovirusesmultiple organ-targeting

Poliovirus--poliomyelitis

EV71--Hand, Foot and

Mouth Disease


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Picornaviridaepico, a small unit of measurement [10-12]RNA, the type of nucleic acid that constitutes the viral genome

EnterovirusPoliovirus

Coxsackievirus

Echovirus

Human enterovirus

Other enteroviruses

Replicate in the human (and other) alimentary tract

(e.g. polio virus)

Rhinovirus Disease of the nasopharyngeal region (e.g. common cold virus)

Cardiovirus Murine encephalomyocardit is, Theiler's murine encephalomyelitisvirus

Aphthovirus Foot and mouth disease in cloven footed animals

Hepatovirus Human hepatitis virus A

Others Drosophila C virus, equine rhinoviruses, cricket paralysis virus


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Properties of enteroviruses

Property Enteroviruses

Size (nm)

Capsid

form

Polypeptide

Genome

Acid, disinfectantsThermostable

UV light

22-30

Icosahedral

VP1, VP2, VP3, VP4(60 Copies each)

ss (+) RNA

StableInactivated at 42

Sensitive


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Enterovirusesmultiple organ-targeting


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Clinical syndromes associated with

infections by enterovirusesPolioviruses,

types1-3

Paralysis (complete to slight muscle weakness)Aseptic

meningitisUndifferentiated febrile illness

Coxsackieviruses,

group A, types 1-24

HerpanginaAcute lymphatic or nodular pharyngitis

ParalysisAseptic meningitisHand-foot-and-mouth disease(A10, A16)Pneumonitis of infantsCommon cold

HepatitisInfantile diarrheaAcute hemorrhagicconjunctivitis(type A24 variant)Exanthema

Coxsackieviruses,

group B, types 1-6

PleurodyniaAseptic meningitisParalysis (infrequently)Severe systemic infection in infants, meningoencephalitis, andmyocarditisPericarditisUpper respiratory illness andpneumoniaRash Hepatitis Undifferentiated febrile illness

Echoviruses,

types 1-33

Aseptic meningitisParalysisEncephalitis, ataxia

ExanthemaRespiratory disease Pericarditis andmyocarditisDiarrheaHepatic disturbance

Enterovirus,

types 68-71

Pneumonia and bronchiolitisAcute hemorrhagic conjunctivitis

(type 70)Paralysis (types 70, 71)Meningoencephalitis(types 70, 71)Hand-foot-and-mouth disease (type 71)


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Although most enterovirus (EV) infections areasymptomatic, estimates are that as many as 5 to 10 millionsymptomatic EV infections occur each year in the UnitedStates.

Most EV infections do not cause significant disease, butinfection can lead to serious illness, especially in infants andin those who are immunocompromised.


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Enteroviruses have been implicated inboth chronic and acute diseases.

Chronic diseases include:

dermatomyositis,

polymyositis,

dilated cardiomyopathy,

diabetes mellitus.


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Transmission


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Immune Response The efficacy of the immune response is key to the

outcome of enteroviral infections.

The innate immune system is especiallyimportant because it is the earliest response and,in addition, it regulates the adaptive immune

response. The humoral immune response in enteroviral

infections is especially important for protection andlife-long immunity.

Cell-mediated immunity is not usually involved inprotection but may play a role in pathogenesis.


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Laboratory Diagnosis

Molecular Detection

Using these guidelines, strains of homologous serotypes can be easilydiscriminated from heterologous serotypes and new serotypes can be identified. Thismethod can greatly reduce the time required to type an EV isolate, and can be usedto type isolates that are difficult or impossible to type using standard immunologicreagents. The technique is also useful to rapidly determine whether viruses isolatedduring an outbreak are epidemiologically related.


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Laboratory Diagnosis

Virus Isolation

Isolation of EV from specimens using appropriate cultured cell lines

is often possible within 2 or 3 days and remains a very sensitivemethod for detecting these viruses.

The best specimens for isolation of virus are, in order of preference,stool specimens or rectal swabs, throat swabs or washings, and

cerebrospinal fluid.


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Cytopathic effect typical of PV infection


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Laboratory DiagnosisAntibody Tests

Serologic diagnosis of EV infection can be made by comparingtiters in acute and convalescent phase (paired) serum

specimens.The most basic serologic test is that of neutralization in cellculture. A fourfold or greater rise in type-specific neutralizingantibody titer is considered diagnostic of infection.

Many serologic studies rely on the detection of IgM antibodyas evidence for recent EV infection, and this is now widelyused as an alternative to the neutralization and complement-

fixation test.In general, however, EV serodiagnosis is more relevant toepidemiologic studies than to clinical diagnosis.


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TreatmentNo specific antiviral agent is available for

therapy of enterovirus infection.Treatment focuses on management ofcomplications (for example, meningitis,

abnormal cardiac rhythms, and heart failure).Intravenous administration of immune globulinmay have a use in preventing severe disease

in immunocompromised individuals or thosewith life-threatening disease.


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Prevention and controlImproved sanitation andgeneral hygiene

The viruses are resistant tomany disinfectants so it isimportant to use chlorinated(bleach) or iodized disinfectants.

During recognised epidemics,it may be advised to closecertain institutions such asschools or child care facilities in

order to reduce transmissionespecially among youngchildren.

It is not necessary to restricttravel or trade.


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Poliovirus


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The oldest clearly identifiable

reference to paralytic poliomyelitis isan Egyptian stele (stone engraving)over 3,000 years old.

In 1840, von Heine published amonograph more specificallydescribing the affliction. Hiscontributions and those publishedlater by Medin from Sweden led toparalytic poliomyelitis being referredto as Heine-Medin disease.


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The 1900s began anew era inpoliomyelitis

investigations, andthe beginning of anunderstanding of theinfectious nature of

this disease:

Wickman I. et.al,1907.

Landsteiner K,Popper E. 1909

Trask JD, et.al,

1938


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In 1945, Burnet wrote, The practical problem of preventing

infantile paralysis has not been solved. It is even doubtfulwhether it ever will be solved.

Despite this progress, a number of unfortunatemisconceptions emerged about poliomyelitis that initiallyconfused scientists and misdirected efforts for control.

These misconceptions included a belief:

that the virus was exclusively neurotropic,

that the nasopharynx was a major site for virus entry into theCNS,

and that the virus spread to the nervous system beforeviremia and by way of the olfactory nerve.

Only the fear surrounding AIDS can rival the feelings people

had about polio in the first half of this century!


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Building on studies of others, Enders et al. (1949) performeda landmark study showing that PV could be propagated innon-neural tissue culture.

A variety of vaccines were subsequently produced, with themost well-known being the Salk inactivated polio vaccine (IPV)delivered via the intramuscular route (licensed in 1955 in theUnited States) and the Sabin live, attenuated vaccine (oral

polio vaccine [OPV]) delivered via the oral route (licensed in1961-1962).


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Model of PV entry

Imaging Poliovirus Entry in Live Cells. PLoS Biol. 2007


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It was known that mouse cells arenot susceptible to poliovirusinfection because they lack cellularreceptors.

Mouse cells are permissive topoliovirus replication, becauseintroduction of poliovirus RNA intothese cells leads to the synthesis

of infectious viruses.Poliovirus-susceptible mousecells were produced by introducinghuman genomic DNA into mouse

cells.

The poliovirus receptor

The poliovirus receptor (PVR, CD155) was identified by DNAtransformation.


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VPg is not required for infectivity of poliovirus RNA.

VPg is not found on viral mRNA that is associated withcellular ribosomes and undergoing translation.

VPg is present on nascent RNA chains of the replicativeintermediate RNA and on negative-stranded RNA.

VPg is a primer for poliovirus RNA synthesis.

Genome Structure and Organization

Paul AV, van Boom JH, Filippov D, et al. Protein-primed RNA synthesis bypurified poliovirus RNA polymerase. Nature 1998;393:280-284.


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The 5-noncoding region is longand highly structured.

This region of the genome containssequences that control genomereplication and translation.

The 5-noncoding region containsthe internal ribosome entry site(IRES) that directs translation of themRNA by internal ribosome binding.


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The 3-noncoding region of picornaviruses is short, and this

region also contains secondary structure, including apseudoknot, that has been implicated in controlling viral RNAsynthesis.

The entire 3-noncoding region of poliovirus is not required,

however, for infectivity.

Both poliovirus virion RNA and mRNA contain a 3 stretch ofpoly(A), viral RNA from which the poly(A) tract is removed is

noninfectious.


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Overview of the poliovirus replication cycle


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Model for the switch between translation

and (-) strand RNA synthesis.

Viral proteins 3CDpro, VPg, PABP,and PCBP interact with each

other and the ends of the viralRNA to form a circular RNPcomplex. Inhibition of translationinitiation clears the viral RNA oftranslating ribosomes (top).

When the template RNA iscleared of ribosomes, VPg-pUpUassociates with the 3end of theviral RNA and forms the circularpreinitiation RNA replicationcomplex (middle).

Negative-strand synthesisinitiates by the elongation of theVPg-pUpU by 3Dpol (bottom).


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Recombination

Recombination, the exchange of nucleotide sequences amongdifferent genome RNA molecules, was first discovered in cellsinfected with poliovirus, and was subsequently found to occurduring infection with other positive- and negative-stranded RNA

viruses.

Poliovirus recombination, whichmainly occurs between nucleotidesequences of the two parentalgenome RNA strands that have a highpercentage of nucleotide identity, iscalled base pairing dependent.

RNA recombination is believed to becoupled with the process of genomeRNA replication: it occurs by template

switching during negative strandsynthesis.

The recombination frequency was0.9%, leading to the estimation that10% to 20% of the viral genomes

recombine in one growth cycle.


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Assembly of Virus Particles

Morphogenesis of picornaviruses hasbeen studied extensively because the60-subunit capsid is relatively simple

and the assembly intermediates can bereadily detected in infected cells.

The capsid protein precursor, P1, iscleaved to VP0 + VP3 + VP1. Protomers

(5S) self-assemble into 14S pentamers,and pentamers assemble into 80S emptycapsids. In one model of encapsidation,RNA is threaded into the empty capsid,producing a 150S provirion in which VP0

is uncleaved. Another possibility is thatpentamers assemble around the RNAgenome and that empty capsids arestorage depots for pentamers. Cleavage ofVP0 is the final morphogenetic step that

produces the infectious 160S virion.


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Poliovirus work has had a continuing significant impact on thefield of molecular virology, many of the PV milestones arelandmarks in the study of EV and, in fact, all of virology.

The discovery in 1949 that poliovirus could be propagated in cultured

cells led to studies of viral replication.The plaque assay, an essential method for quantification of viralinfectivity, was developed with poliovirus (1952).The synthesis of a precursor polyprotein from which viral proteinsare derived by proteolytic processing was first identified in poliovirus-infected cells (1968).Poliovirus RNA was the first messenger RNA (mRNA) shown to lacka 5 cap structure (1976).PV was the first animal virus completely cloned and sequenced

(1981), the first RNA animal virus for which an infectious clone wasconstructed (1981), and the first human virus that had its three-dimensional structure solved by x-ray crystallography(1985).

Poliovirus is truly a virus with a brilliant past, but with no future.


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Non-Polio Enterovirus


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Coxsackieviruses Coxsackieviruses are distinguished from other enteroviruses by their

pathogenicity for suckling rather than adult mice. They are dividedinto 2 groups on the basis of the lesions observed in suckling mice.

Group A viruses produce a myositis with flaccid hind limbparalysis in newborn mice.

Group B viruses produce produced a spastic paralysis and

generalized infection in newborn mice, with myositis as well asinvolvement of the brain, pancreas, heart, and brown fat.

It is believed that 3.2% of CVB infections, result in overt cardiacsigns or symptoms. A prior history of enteroviral infection of theheart, especially with CV B, has been implicated in sporadic human

dilated cardiomyopathy.

Coxsackieviruses, especially CVB4, have been implicated in thecause of insulin-dependent diabetes.


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Echoviruses The first echoviruses were accidentally discovered in human faeces,

unassociated with human disease during epidemiological studies of

polioviruses(1951). Echoviruses received their name because theywere enteric isolates, cytopathogenic in tissue culture, isolated fromhumans, and orphans (i.e., unassociated with a known clinicaldisease).

These viruses were produced CPE in cell cultures, but did not inducedetectable pathological lesions in suckling mice.

Subsequent studies have shown that echoviruses, in fact, do cause avariety of human diseases( Aseptic meningitis;Paralysis;Encephalitis,ataxia;Exanthema;Diarrhea;Respiratory disease;Pericarditis and

myocarditis;et.al ).

Altogether, there are 32 echoviruses (types 1-34; echovirus 10 and 28were found to be other viruses and thus the numbers are unused).


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New Enteroviruses Newly identified enteroviruses that are not polioviruses are no

longer classified separated into the species coxsackie andechovirus. 4 new enteroviruses have been identified (68 - 71).

Enterovirus 70 is the causative agent epidemics of acutehaemorrhagic conjunctivitis that swept through Africa, Asia,

India and Europe from 1969 to 1974. The virus is occasionallyneurovirulent.

Enterovirus 71 appears to be highly pathogenic and has beenassociated with epidemics of a variety of acute diseases,

including aseptic meningitis, encephalitis, paralyticpoliomyelitis-like disease and hand-foot-mouth disease.

Enterovirus 72 was originally assigned to hepatitis A virus, butit had now been assigned to a new family called heptoviruses.


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Hepatitis A Virus


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Ancient Chinese literature referred to theoccurrence of jaundice, as reviewed byZuckerman, as did a letter from Pope Zacharias

to Archbishop St. Boniface of Mainz during the8th century AD.

In 1923, Blumer summarized the pattern ofillness found in 63 outbreaks of epidemic

jaundice reported in the United States between1812 and 1922.

In 1947, MacCallum introduced the termshepatitis A and hepatitis B to categorize these

diseases.

1979, Provost and Hilleman were successful incultivating and serially passaging HAV in cell

culture.


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HAV differs from other picornaviruses in that:

(a) nucleotide and amino acid sequences and thepredicted sizes of several HAV proteins are dissimilar;

(b) HAV replicates very slowly without cytopathic effect incell culture;

(c) HAV is resistant to temperatures and drugs that

inactivate many picornaviruses;

(d) HAV is stable at pH 1;

(e) HAV has only one serotype, and one neutralization

site is immunodominant;

(f) an enterovirus-specific monoclonal antibody does notreact with HAV.


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Management of Acute Viral Hepatitis

No specific treatment for acute viral hepatitis exists.

Therapy should be supportive and aimed at maintaining

comfort and adequate nutritional balance.For most jaundiced patients, strict bed rest and prolongedconfinement are probably not indicated.

Prevention and Control

The most critical control measure is proper handwashing,and the avoidance of work practices that facilitatecontamination of hands when taking care of children youngerthan 2 years.


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Questions:

Please give the reasons why poliovirus has

provided so much information in the field ofmolecular biology.

Use poliovirus as an example to discuss the

factors which might be involved in virustropism.

What did you learn from the story of

poliovirus? Will HIV be the next one?


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References Crowder S, Kirkegaard K. Trans-dominant inhibition of RNA viral

replication can slow growth of drug-resistant viruses. Nat Genet2005;37:701-09.

Duintjer Tebbens RJ, Pallansch MA, Kew OM, et al. A dynamic model ofpoliomyelitis outbreaks: learning from the past to help inform the future.Am J Epidemiol 2005;162(4):358-72.

Gitlin L, Stone, JK, Andino, R. Polivirus escape from RNA interference:short interfering RNA-target recognition and implications for therapeuticapproaches. J Virol 2005;79:1027-035.

Heymann DL, Sutter RW, Aylward RB. A global call for new polio vaccines.Nature 2005;434:6099-000.

Mueller S, Cao X, Welker R, et al. Interaction of the poliovirus receptor

CD155 with the dynein light chain Tctex-1 and its implication forpoliovirus pathogenesis. J Biol Chem 2002;277:7897-04.

Coyne CB, Bergelson JM. Virus-induced Abl and Fyn kinase signalspermit coxsackievirus entry through epithelial tight junctions. Cell2006;124:119-31.


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