PICORNAVIRIDAE

Dr. R. Someshwaran, MD., Assistant professor, Department of Microbiology, KFMS&R

Objectives of today’s classTo know and understand the morphology, classification, pathogenesis of Picornaviruses

To learn about the clinical manifestations, lab diagnosis, treatment, prevention and prophylaxis of Poliomyelitis.

To learn about the clinical manifestations, lab diagnosis, treatment, prevention and prophylaxis of ECHO and Rhinoviruses.

Introduction to PicornavirusesFamily Picornaviridae Consists of a large number of very small RNA viruses,

27-30 nm in sizeResistant to lipid solvents like Ether, Chloroform, Bile salts

Morphology of Enterovirus

Morphology of Poliovirus Electron microscopy (EM) of Poliovirus

Epidemiology

Epidemiology

Classification• 4 Genera pathogenic to humans:1. Enterovirus – Infects intestinal tract2. Rhinovirus – infects nasal mucosa3. Hepatovirus 4. Paraechovirus• 2 more Genera pathogenic for animals1. Aphthovirus – Foot and Mouth disease of catlle2. Cardiovirus – Encephalomyocarditis virus that infects

mice

ENTEROVIRUSES - History• Infantile paralysis – Paralytic disease of children• Landsteiner and Popper (1909) demonstratedFatal case of Poliomyelitis – Spinal cord & Feces inoculation – Experimental disease transmission - monkeys

ENTEROVIRUSES – History Continued

• Enders, Wellers & Robbins (1949) demonstrated• Growth of Poliovirus • Culture of non-neural cells from human embryos• Produced Cytopathic effects (CPE)• Major break through – Nobel prize for this discovery • Development of Virology (Milestone)

ENTEROVIRUSES – History Continued….

• Dalldorf & Sickles (1948) demonstration:• A new type of virus isolated• From feces of children with paralytic poliomyelitis• Named Type 1 Poliovirus • This virus caused paralysis on inoculation in suckling

mice• Was called the Coxsackie virus • As patient was from village ‘Coxsackie’ in New York

ENTEROVIRUSES – History Continued ………

• Introduction of Tissue culture technique• Diagnostic virology • Led to Fecal isolation of several cytopathogenic viruses

from cases• Were called ‘Orphan viruses’• Not associated with any particular clinical disease• Was known by descriptive term “ECHO” • ECHO – Entero cytopathogenic human orphan viruses

ENTEROVIRUSES – Characteristics• Entero viruses are ‘Host specific’• Infects only one or a few related species• No common group antigen • Antigen cross reactions observed in few

ENTEROVIRUSES – Classification Group Serotype

Poliovirus (3) Type 1, Type 2, Type 3Coxsackie virus A (24) 1-22, 24Coxsackie virus (6) 1-6Echovirus (34) 1-9, 11-27, 29-31Numbered enterovirus(EV since 1969 – Numbered 68 and so)

68-71

Poliovirus• Clinical case scenarion:• A 12 years old boy from Kovilpalayam presented to the

emergency department of KFMS&R with a h/o mild fever and sore throat X 8 days, condition worsened and became severe accompanied by neck rigidity and vomiting for last 2 days after a brief asymptomatic period of 2 days.• HOPI: On morning, boy experienced pain in lower limbs which

increased and progressed as weakness at the time of presentation.• Mother’s history: Childhood (<5 years) vaccination not

remembered

Poliovirus•Diagnostic & Management strategy of Case scenarion:• Throat swab, Stool, CSF sent to Diagnostic Microbiology

Dept. for viral studies• Throat swab and stool specimen tested positive for Cytopathic effect (CPE) in tissue culture.• Virus was confirmed as Poliovirus type 1 by Neutralization test• Reverse Transcriptase PCR was positive for Poliovirus in

CSF• Patient improved on supportive treatment.

Poliovirus - Introduction• Group IV; Family Picornaviridae; Genus Enterovirus; Species Poliovirus• The virus is composed of an RNA genome and a protein

capsule. The genome is single-stranded positive-sense RNA genome that is about 7500 nucleotides long. • Often called the simplest significant virus - First isolated

in 1909 by Karl Landsteiner and Erwin Popper • Egyptian paintings depicted the effects of polio by

showing otherwise healthy individuals with withered limbs.

Morphology• Spherical Virion – Icosahedral symmetry• 27 nm in diameter in size• Composed of 60 subunits• Consists of 4 Viral Proteins (VP1, VP2, VP3 & VP4)• VP1 faces outside – Major antigenic site for combination• VP1 has type-specific neutralizing antibodies• Viral Genome: Single stranded positive strand (ss RNA

+ve sense)• Virus can be crystallized – seen in cytoplasm of infected

cells

Resistance• Resistant to lipid solvents – ether, chloroform, bile, proteolytic

enzymes of intestinal contents and detergents• Stable at a pH of 3• In feces, it can survive for 4 months at 4ºC and for years at -20ºC• Room temperature survival of virus in feces vary (one day to

several weeks) and It depends on temperature, moisture, pH and amount of virus• Readily inactivated by heat (55ºC X 30 minutes)• Molar MgCl2 , Milk or Icecream protects virus against heat

inactivation

Resistance• Formaldehyde and Oxidising disinfectants destroy the

virus• Chlorination destroy the virus in water• Organic matter present delays inactivation of virus• Phenolic disinfectants not effective• Does not survive lyophilisation well

Antigenic properties• Based on Neutralisation test• Poliovirus (PV) classified into 3 types: Type 1, Type 2 &

Type 3• Prototype strains: Brunhilde & Mahoney strains for Type 1Lansing and MEFI for Type 2Leon and Saukett for Type 3Type 1 Most common, causes most

epidemicsType 2 Usually cause endemic diseaseType 3 Strains cause epidemic.

Antigenic properties• Based on Complement Fixation Test (CFT) or Enzyme Linked Immuno Sorbent Assay (ELISA) or Precipitation tests• Two antigens recognized: C & D1. C antigen - Capsid, Coreless, also called Heated or H

antigen2. D antigen – Dense, also called Native or N antigen • D antigen / N antigen associated with whole virion – Type specific• C antigen / H antigen associated with ‘empty’ non-infectious

virus and Less specific and reacts with heterotypic sera

Antigenic properties• D antigen – converted to C antigen by heating the virus

at 56⁰C• Anti D antibody is protective. So, Potency of Injectable

Polio Vaccine can be measured in terms of D antigen units.• Anti-C antibody does not neutralize virus infectivity

Host range & Cultivation• Natural infection occurs only in humans• Experimental transmission in monkeys by intracerebral

or intraspinal inoculation• Chimpanzees and Cynomolgus monkeys – can be

infected orally• Established non-fresh strains can be grown in rodents,

chick embryos• Virus grows readily in Tissue cultures of primate origin• Primary Monkey Kidney cultures are used for Diagnostic

purpose and for Vaccination

Host range & Cultivation• Cytopathic effect (CPE): Infected cells round up and

become refractile and pyknotic. • Eosinophilic intranuclear inclusion bodies – may be

demonstrated in stained preparations.• Well-formed plaques develop in infected monolayers

with agar overlay.

Pathogenesis• The polio virus infects human cells by binding to an

immunoglobubin-like receptor called CD155 (poliovirus receptor). • The exact mechanism that poliovirus uses for entering the cell is

unknown. • However, the interaction of poliovirus and CD155 causes a change

in the shape of the viral particle that is needed to enter the cell• There are two thesis' for the way the viral nucleic acid to enters

the cell. 1. RNA of poliovirus is injected into the host cell through a

pore in the membrane of the host cell. 2. The poliovirus is taken in by the host cell through

endocytosis.

Pathogenesis• The genome inside poliovirus can be used as mRNA and immediately translated by the host cell. • The poliovirus mRNA is then translated into a long

polypeptide which is cleaved into 10 individual viral proteins. • Translation of the viral RNA occurs by an IRES-mediated (internal ribosome entry site) mechanism. The IRES is the extremely long 5’ end of the poliovirus’ mRNA. The assembly of viral particles is not fully understood.• The particles leave the host cell 4-6 hours after the initial

infection. Each dying host cell can release 10,000 polio virions making poliovirus lytic.

Pathogenicity1. Virus transmitted by Fecal-oral route through

ingestion. Other possible modes in close contacts in patients of early stages: Inhalation or Entry through conjunctiva of droplets of respiratory secretions.

2. Virus multiplies initially in the epithelial cells of the alimentary canal and in the lymphatic tissues, from the tonsils to peyer’s patches

3. Spreads to regional lymph nodes and enters blood stream (Primary viremia)

4. Further multiplication takes place in reticulo-endothelial system

Pathogenesis

Pathogenicity - Continues5. Virus enters the Blood stream again (Secondary viremia)6. Virus is now carried to Central Nervous System (CNS) Spinal

cord and Brain. 7. In CNS, Virus multiplies in selective neurons and destroys them8. Earliest change: Degeneration of Nissl’s or bodies

(Chromatolysis)9. Nuclear changes follows.10.When degeneration becomes irreversible, the necrotic cells

lyses or is phagocytosed by leucocytes or macrophages

Pathogenicity - Continued11.Lesions are mostly in the anterior horns of the spinal cord

causing Flaccid paralysis, but posterior and intermediate horns can also be involved.

12.Pathological changes will be more than distribution of paralysis13.Encephalitis primarily involving the brain stem and extending

up to motor and pre-motor areas can occur in some cases.14.Special circumstances: Direct neural transmission of Virus to

Central Nervous System (CNS) as in poliomyelitis following Tonsillectomy

15.Poliomyelitis: Polio= gray matter; Myelitis= Spinal cord inflammation

Clinical featuresPoliomyelitis, or polio, is a crippling disease caused byany one of three related viruses, poliovirus types 1, 2 or 3.

1.Inapparent infection2.Minor illness3.Paralytic poiliomyelitis or Major

illness

Clinical featuresInapparent infection: 90-95% susceptible individuals develop only inapparent polio

infection with Seroconversion alone.Only 5-10% among them develop clinical infection. Incubation

period: About 10 days on average (Range:4 days – 4weeks)Minor illness: Early manifestation is fever, headache, sore throat and malaise

(Phase of primary viremia) lasting 1-5 days called Minor illness or Abortive poliomyelitis

Paralytic poliomyelitis or major illness: Progression of infection 3-4 dyas after minor illness results in

major illness. Fever returns (Biphasic fever), along with headache, stiff neck and other features of meningitis due to viral invasion of CNS (Polio case).

Pathogenesis

Poliomyelitis types and Complication

• Non- Paralytic polio: Disease does not progress beyond stage of aseptic meningitis• So, Types of polio: Nonparalytic polio & Paralytic polio. • Paralytic polio can further be divided into: Spinal polio,

bulbar polio, and bulbospinal polio based on distribution of paralysis.• Complication: Post-polio syndrome may also occur in which

symptoms ranging form breathing and swallowing problems to joint pain, start many decades after the initial sickeness.• Vaccine associated Poliomyelitis

Laboratory diagnosis• Samples to be collected: Blood, CSF, Throat swab, Feces• Transport: Immediately to lab in viral transport media

(Hank’s Balanced Salt Solution)• Storage: 4⁰C (Days), -20⁰C (months to years)• Laboratory Diagnostic tests available:Viral isolationSerodiagnosisMolecular diagnosis

1. Viral isolation• In tissue culture – during primary Viremia 3-5 days after

infection before neutralizing antibodies appear (from blood)• Early stages – isolation from throat swabs• First week of infection: 80-85% viral isolation from feces• Second week of infection: 50% viral isolation from feces• Third week of infection: 25% viral isolation from feces• Fecal excretion – intermittent (So two samples needed to be

tested)• Prolonged fecal excretion in immunocompromised, but no

permanent carriers,

1. Viral isolation - continues• Seldom poliovirus isolated from CSF, but it can be

isolated from spinal cord and brain, post-mortem diagnosis. (Unlike enteroviruses)• Primary monkey kidney cells are employed commonly• Human or Simian kidney cells can also be used.• Inference: Viral growth indicated by Cytopathic effect (CPE) in 2-3 days.• Mere isolation does not confirm the diagnosis

2. Serodiagnosis • Antibody rise appears after the onset of paralysis – demonstrated by Neutralisation tests or Complement Fixation Tests (CFT). • Neutralising antibodies appear early and persista for life• In CFT, Anti-C antibodies appear first and disappear in few months.• Anti-D antibodies rise after few weeks and lasts for 5 years.•CFT is useful to identify the exposure to Poliovirus but not for type-specific diagnosis

3. Molecular diagnosis • Reverse Transcriptase PCR: Viral RNA in CSF demonstrated• RNA Sequencing: In circulation 3 types of strains circulate

1. Wild virus2. OPV strain (Oral polio Virus)3. VDPV – Vaccine Derived Polio Virus

Immunity• Type-specific immunity• Humoral immunity – circulating and secretory antibodies responsible

for protection against Poliomyelitis.• IgM – appear in 1 week and lasts for 6 months• IgG – appear by 6 months and persists for life• Neutralising antibodies protects against disease• Secretory IgA – in GIT – provides Intestinal immunity.

Immuno-prophylaxis

Immuno-prophylaxis

Live oral polio vaccine (OPV) - four doses in endemic countriesor Inactivated polio vaccine (IPV) given by injection - two-three doses depending on country schedule

Immuno-prophylaxis

Immuno-prophylaxis

Immuno-prophylaxis• UIP: Both IPV and OPV are safe• Salk (IPV) – Killed formolised, contains type1, 2 & 3, S.c or Im• Sabin (OPV) – Live attenuated vaccine grown in Monkey kidney cells• OPV has Type 1 virus 10 lakh, type 2 virus 2 lakh, Type 3 virus 3 lakh• TC ID50 per dose (0.5mL) – vaccine maintained in air-tight container• Pulse Polio Immunisation• Local Intestinal immunity• Herd Immunity

Treatment• Currently there is no treatment to cure Polio. Treatment

is focused on supportive care. Moderate exercise - A nutritious diet • Medication and rest to lower the fever and to reduce the pain

and improve the strength. Breathing assistance with a ventilator• To Prevent Poliovirus: The most effective and most commonly

used is the Polio vaccine. This vaccine is given to young children in specific increments. • Vaccine works by strengthening and preparing the immune

system to a future encounter with the Poliovirus.

Coxsackie Group A virus• 24 serotypes• Causes:1. Herpangina (Vesicular pharyngitis), 2. Hand, Foot and Mouth disease (HFMD), 3. Aseptic meningitis, 4. Minor respiratory infection

Coxsackie Group B virus• 6 serotypes• Causes:1. Epidemic pleurodynia or Bornholm disease2. Myocarditis and pericarditis3. Juvenile Diabetes – Coxsackie B4 ?4. Orchitis5. Transplacental and neonatal transmission6. Post-viral fatigue syndrome

Coxsackie virus• Lab diagnosis:1. Animal inoculation – inoculating in suckling mice2. Tissue culture – not useful3. Serodiagnosis not practicable – due to several antigenic types

ECHO virusEnterocytopathogenic Human orphan virusGrow in Human and simian kidney cultureFever with rash, Aseptic Meningitis (most common cause)Lab diagnosis: Feces, throat swab, CSF - Culture

New Entero virusAcute hemorrhagic conjunctivitis – EV 68,69,70,71Radiculopathy – EV-70Grows in Human embryonic kidney or HeLa cell lines.

RhinovirusesCommon cold virus – 100 serotypes by neutralizationTransmitted by droplet infectionCulture – Just like for ECHO and other new entero viruses

Summary• Polio virus – Belong to Entero virus – ss RNA virus +

sense• Polio virus: 3 types – 1, 2 & 3• CD 155 receptor – Feco-oral transmission• Paralytic or Non –paralytic polio• CSF, Blood, Throat swab & feces• Neutralisation tests, ELISA, CFT, RT- PCR• OPV, IPV –Salk and Sabin• Pulse polio immunization programme.

Take home message

OPV and IPV to chuck out Polio completely

Characteristics of Poliovirus

Poliovirus is an Entero virus. True or False?

Poliovirus is a RNA virus. True or False?

Poliovirus is transmitted by droplet nuclei. True or False?

Salk vaccine is a Killed vaccine. True or False?

Sabin vaccine is a Live attenuated vaccine. True or False?

Oral Polio Vaccine is given as single dose at birth. True or False?

References• Ananthanarayan & Paniker’s Textbook of Microbiology,

9th Edition• NAMH. How Polio Works.

http://polio.emedtv.com/polio/polio-treatment.html• MayoClinic.com. Infectious Disease.

http://www.mayoclinic.com/health/polio/DS00572/DSECTION=symptoms• Wikipedia. Poliovirus.

http://en.wikipedia.org/wiki/Poliovirus• Wikipedia. Poliomyelitis.

http://en.wikipedia.org/wiki/Poliomyelitis

THANK YOU