Picorna viruses
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This document provides an overview of picornaviruses, with a focus on poliovirus. It begins by outlining the objectives of discussing picornavirus morphology, classification, pathogenesis, and the clinical manifestations, diagnosis, treatment and prevention of poliomyelitis and other diseases caused by echoviruses and rhinoviruses. It then provides details on picornavirus morphology, classification, the history of poliovirus research and discovery, epidemiology, characteristics, cultivation, pathogenesis, and clinical features of poliomyelitis. Key points covered include that poliovirus is an enterovirus that can cause paralysis, replication in the gastrointestinal tract and nervous system, and that infection may be asymptomatic, minor
Picorna viruses
- 1. PICORNAVIRIDAE Dr.DineshKr Jain, MD., Assistantprofessor, Department of Microbiology,SMS Medical college, Jaipur
- 2. Objectives of today’s class Toknow and understand the morphology,classification, pathogenesis of Picornaviruses Tolearn about the clinical manifestations, lab diagnosis, treatment, prevention and prophylaxis ofPoliomyelitis. Tolearn about the clinical manifestations, lab diagnosis, treatment, prevention and prophylaxis of ECHOand Rhinoviruses.
- 3. Introduction to Picornaviruses Family Picornaviridae Consists of a large number of very small RNAviruses, 27-30 nm insize Resistant to lipid solvents like Ether, Chloroform, Bile salts
- 6. Epidemiology
- 7. Epidemiology
- 8. Classification • 4 Genera pathogenic to humans: 1. Enterovirus – Infects intestinal tract 2. Rhinovirus – infects nasal mucosa 3. Hepatovirus 4. Paraechovirus • 2 more Genera pathogenic for animals 1. Aphthovirus – Foot and Mouth disease of catlle 2. Cardiovirus – Encephalomyocarditis virus that infects mice
- 9. ENTEROVIRUSES- History • Infantile paralysis – Paralytic disease of children • Landsteiner and Popper (1909) demonstrated Fatal case of Poliomyelitis – Spinal cord &Feces inoculation – Experimental disease transmission - monkeys
- 10. 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)
- 11. ENTEROVIRUSES– History Continued…. • Dalldorf &Sickles (1948) demonstration: • Anew 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 • Aspatient was from village ‘Coxsackie’in NewYork
- 12. 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
- 13. 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
- 14. ENTEROVIRUSES– Classification Group Serotype Poliovirus (3) Type 1, Type 2, Type 3 Coxsackie virus A(24) 1-22, 24 Coxsackie virus (6) 1-6 Echovirus (34) 1-9, 11-27, 29-31 Numbered enterovirus (EVsince 1969 – Numbered 68 andso) 68-71
- 15. Poliovirus • Clinical case scenarion: • A12 years old boy from Kovilpalayam presented to the emergency department of KFMS&Rwith a h/o mild fever and sore throat X8 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
- 16. Poliovirus • Diagnostic &Management strategy of Case scenarion: • Throat swab, Stool, CSFsent to Diagnostic Microbiology Dept. for viral studies • Throat swab and stool specimen tested positive for Cytopathic effect (CPE)in tissueculture. • Virus was confirmed as Poliovirus type 1 by Neutralization test • ReverseTranscriptase PCRwas positive for Poliovirus in CSF • Patient improved on supportive treatment.
- 17. Poliovirus - Introduction • Group IV;FamilyPicornaviridae; Genus Enterovirus; Species Poliovirus • The virus is composed of an RNAgenome 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 1909by Karl Landsteiner and Erwin Popper • Egyptian paintings depicted the effects of polio by showing otherwise healthy individuals with withered limbs.
- 18. Morphology • Spherical Virion – Icosahedral symmetry • 27 nm in diameter insize • Composed of 60 subunits • Consists of 4 Viral Proteins (VP1,VP2,VP3 &VP4) • VP1 faces outside – Major antigenic site for combination • VP1has type-specific neutralizing antibodies • Viral Genome: Single stranded positive strand (ss RNA+ve sense) • Virus can be crystallized – seen in cytoplasm of infected cells
- 19. 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 X30 minutes) • Molar MgCl2 , Milk or Icecream protects virus against heat inactivation
- 20. 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
- 21. Antigenic properties • Based on Neutralisation test • Poliovirus (PV)classified into 3 types: Type 1, Type 2 &Type 3 • Prototype strains: Brunhilde &Mahoney strains for Type1 Lansing and MEFIfor Type2 Leon and Saukett for Type 3 Type 1 Most common, causes most epidemics Type 2 Usually cause endemic disease Type 3 Strains causeepidemic.
- 22. Antigenic properties • Based on Complement Fixation Test(CFT)or Enzyme Linked Immuno SorbentAssay (ELISA)or Precipitation tests • Two antigens recognized: C&D 1. Cantigen - Capsid, Coreless, also called Heated or Hantigen 2. D antigen – Dense, also called Native or N antigen • D antigen / N antigen associated with whole virion – Type specific • Cantigen / H antigen associated with ‘empty’non-infectious virus and Less specific and reacts with heterotypic sera
- 23. Antigenic properties • D antigen – converted to Cantigen 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
- 24. 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
- 25. 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.
- 26. 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 thecell • There are two thesis' for the way the viral nucleic acid to enters the cell. 1. RNAof poliovirus is injected into the host cell through a pore in the membrane of the host cell. 2. Thepoliovirus is taken in by the host cell through endocytosis.
- 27. Pathogenesis • Thegenome inside poliovirus can be used as mRNAand immediately translated by the host cell. • The poliovirus mRNAis then translated into a long polypeptide which is cleaved into 10 individual viral proteins. • Translation of the viral RNAoccurs 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 initialinfection. Each dying host cell can release 10,000 polio virions making poliovirus lytic.
- 28. Pathogenicity 1. 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
- 29. Pathogenesis
- 30. Pathogenicity - Continues 5. 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 them 8. 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
- 31. Pathogenicity - Continued 11. 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 paralysis 13. Encephalitis primarily involving the brain stem and extending up to motor and pre-motor areas can occur insome cases. 14. Special circumstances: Direct neural transmission of Virus to Central Nervous System (CNS) as in poliomyelitis following Tonsillectomy through glossopharyngeal nerve present in tonsillar fossa. 15. Poliomyelitis: Polio= gray matter; Myelitis= Spinal cord inflammation
- 32. Clinical features Poliomyelitis,orpolio,isacripplingdiseasecausedby anyoneofthreerelatedviruses,poliovirustypes1,2or 3. 1. Inapparent infection 2. Minor illness 3. Paralytic poiliomyelitis or Major illness
- 33. Clinical features Inapparent infection: 90-95% susceptible individuals develop only inapparent polioinfection with Seroconversion alone. Only 5-10% among them develop clinical infection. Incubationperiod: About 10 days on average (Range:4 days –4weeks) Minor illness: Early manifestation is fever, headache, sore throat and malaise (Phaseof primary viremia) lasting 1-5days 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).
- 34. Pathogenesis
- 35. 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
- 37. 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(monthsto years) • Laboratory Diagnostic tests available: Viral isolation Serodiagnosis Molecular diagnosis
- 38. 1. Viral isolation • In tissue culture – during primary Viremia 3-5 days afterinfection 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,
- 39. 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
- 40. 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. •CFTis useful to identify the exposure to Poliovirus but not for type-specific diagnosis
- 41. 3. Molecular diagnosis •Reverse Transcriptase PCR: Viral RNAin CSFdemonstrated •RNASequencing: In circulation 3 types of strains circulate 1. Wild virus 2. OPVstrain (Oral polio Virus) 3. VDPV– Vaccine Derived PolioVirus
- 42. Immunity • Type-specificimmunity • Humoralimmunity– circulatingandsecretoryantibodiesresponsible for protectionagainstPoliomyelitis. • IgM– appearin1weekandlastsfor6months • IgG– appearby6monthsandpersistsfor life • Neutralisingantibodiesprotectsagainstdisease • SecretoryIgA– inGIT– providesIntestinalimmunity.
- 44. Immuno-prophylaxis Liveoral polio vaccine (OPV) -four doses in endemiccountries or Inactivated polio vaccine (IPV) given by injection - two-threedoses depending on countryschedule
- 48. Immuno-prophylaxis • UIP:BothIPVandOPVaresafe • Salk(IPV)– Killedformolised,containstype1,2&3,S.cor Im • Sabin(OPV)– LiveattenuatedvaccinegrowninMonkeykidneycells • OPVhasType1virus10lakh,type2virus2lakh,Type3virus3lakh • TCID50perdose(0.5mL)– vaccinemaintainedinair-tightcontainer • PulsePolioImmunisation • LocalIntestinalimmunity • HerdImmunity
- 49. Treatment • Currently there is no treatment to cure Polio. Treatment is focused on supportive care. Moderate exercise - Anutritious diet • Medication and rest to lower the fever and to reduce the painand improve the strength. Breathing assistance with a ventilator • ToPrevent 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.
- 50. Coxsackie Group Avirus • 24 serotypes • Causes: 1. Herpangina (Vesicular pharyngitis), 2. Hand, Foot and Mouth disease (HFMD), 3. Aseptic meningitis, 4. Minor respiratory infection
- 51. Coxsackie Group Bvirus • 6 serotypes • Causes: 1. Epidemic pleurodynia or Bornholm disease 2. Myocarditis and pericarditis 3. Juvenile Diabetes – Coxsackie B4 ? 4. Orchitis 5. Transplacental and neonatal transmission 6. Post-viral fatigue syndrome
- 52. Coxsackie virus • Labdiagnosis: 1. Animal inoculation – inoculating in suckling mice 2. Tissue culture – not useful 3. Serodiagnosis not practicable – due to several antigenic types
- 53. ECHOvirus Enterocytopathogenic Human orphan virus Grow in Human and simian kidney culture Fever with rash, Aseptic Meningitis (most common cause) Lab diagnosis: Feces, throat swab, CSF- Culture
- 54. New Entero virus Acute hemorrhagic conjunctivitis – EV68,69,70,71 Radiculopathy – EV-70 Grows in Human embryonic kidney or HeLa cell lines.
- 55. Rhinoviruses Common cold virus – 100 serotypes by neutralization Transmitted by droplet infection Culture – Just like for ECHO and other new entero viruses
- 56. Summary • Polio virus – Belong to Entero virus – ss RNAvirus +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.
- 58. Characteristics of Poliovirus PoliovirusisanEnterovirus. TrueorFalse? PoliovirusisaRNAvirus. TrueorFalse? Poliovirusistransmittedbydroplet nuclei. TrueorFalse? SalkvaccineisaKilled vaccine. TrueorFalse? SabinvaccineisaLiveattenuated vaccine. TrueorFalse? OralPolioVaccineisgivenassingledoseat birth. TrueorFalse?
- 59. 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=sympt oms • Wikipedia. Poliovirus. http://en.wikipedia.org/wiki/Poliovirus • Wikipedia. Poliomyelitis. http://en.wikipedia.org/wiki/Poliomyelitis
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