1. Viruses associated with respiratory tract infections Medical Virology Lecture 03 Youjun Feng Center for Infection & Immunity, Zhejiang University School of Medicine fengyj@zju.edu.cn

2.  Orthomyxoviridae 正粘病毒 Influenza virus 流感病毒 Avian influenza virus 禽流感病毒 H5N1/H9N2/H7N9  Paramyxoviridae 副粘病毒 Measles virus 麻疹病毒 Mumps virus 腮腺炎病毒 Parainfluenza virus 副流感病毒 Respiratory syncytial virus, RSV 呼吸道合胞病毒  Coronavirus 冠状病毒 & SARS Coronavirus SARS 冠状病毒  Others Adenovirus 腺病毒 Rubella virus 风疹病毒 Rhinovirus 鼻病毒 Reovirus 呼肠病毒

3. envelope Togavirus Orthomyxovirus Influenza virus Paramyxovirus Measles virus Mumps virus Respiratory syncytial virus - ssRNA segmented, Parainfluenza virus - ssRNA, envelope Rubella virus + ssRNA, envelope Picornavirus Rhinovirus Small RNA Viruses Non-enveloped + strand RNA Coronavirus + ssRNA, envelope Adenovirus dsDNA, naked, fibers Reovirus Rotavirus segmented, dsRNA, naked

4. Introduction Orthomyxoviruses (influenza viruses) orthos, Greek for "straight"; myxa, Greek for "mucus" influenza viruse A, B, C influenza viruse A, B—human disease enveloped, segmented negative-sense RNA genome flu-epidemics (local dissemination) or pandemics (worldwide)

5. Flu pendemics recorded

9. Orthomyxoviruses (influenza viruses) Properties Structure and composition

10. 50 nm Structure of influenza virus Virion Spherical, pleomorphic 80-120 nm in diameter

11. Structure of influenza virus HA - hemagglutinin NA - neuraminidase helical nucleocapsid (RNA plus NP protein) lipid bilayer membrane polymerase complex M1 protein RNA

12. 1.Core RNA Composition of influenza virus -ssRNA, 8 segments (type C 7) NP (nucleoprotein) RNA dependent RNA polymerase

13. Composition of influenza virus 2. envelope M protein lipid envelope spike hemagglutinin(HA) neuraminidase(NA) 5151

14. Functions of hemagglutinin & neuraminidase HA causes agglutination of red blood cells. Viruses bind to the mucous membrane cells by HA1 interacting with membrane receptor. Virus’ envelope fuse with cell membrane by HA2 forming a fusion pore. NA help the virus to permeate mucin and escape from “non- specific”inhibitor. NA can increase the number of free virus particles, hence more virus spread from the original site of infection. NA is important in the final stages of release of the new virus particle from infected cells.

16. Classification type A, B, C : NP, M1 protein sub-types: HA or NA protein 16 types HA; 10 types NA NP HA M1 NA

17. Influenza A virus subtypes in the human population

18. Nomenclature Type Host of origin geographical origin strain number antigenic description of HA and NA (parentheses) e.g. A/swine/Iowa/3/70(H1N1) A/Hong Kong/1/68(H3N2)

19. Orthomyxoviruses (influenza viruses) Properties Antigenic drift and antigenic shift

21. Antigenic Drift antigenic drift Minor changes in antigens due to gene mutation in influenza virus. HA and NA accumulate mutations – RNA virus immune response no longer protects fully sporadic outbreaks, limited epidemics

22. Antigenic Drift results in the emergence of dominant strains in the yearly H1N1, H3N2, and strain B

23. Antigenic Shift antigenic shift Major changes in antigens due to gene reassortment in influenza virus. “new” HA or NA proteins pre-existing antibodies do not protect might result in pandemics

24. where do “new” HA and NA come from? 16 types HA 10 types NA – all circulate in birds pigs – avian and human

25. where do “new” HA and NA come from?

26. reassortment

27. Antigenic shift, or reassortment, can result in novel and highly pathogenic strains of human influenza

29. why do we not have influenza B pandemics? so far no shifts have been recorded no animal reservoir known

30. Orthomyxoviruses (influenza viruses) Properties Culture & resistance

31. Culture Chick embryo inoculation Cell culture: PMK, MDCK No obvious Cytopathic effect (CPE) Viral detection depends on RBC agglutination phenomenon HA binds to sialic acid receptor on RBC of human, chicken,guinea pig

32. Resistance Relatively hardy in vitro : maybe stored at 0-4C for weeks Ether and protein denaturants destroy infectively more resistant to alkaline pH than at acid pH (infectivity and hemagglutination)

33. Orthomyxoviruses (influenza viruses) Influenza virus infections in humans Pathogenesis and pathology

34. Before infection DECREASED CLEARANCE RISK BACTERIAL INFECTION (staphylococci, streptococci, haemophilus influenzae) VIREMIA RARE Viral NA lowers the viscosity of the mucous film in the respiratory tract

35. By person-to-person spread through respiratory droplets. Transmission of Influenza Virus AEROSOL – 100,000 TO 1,000,000 VIRIONS PER DROPLET 1-4 days INCUBATION SHEDDING

36. Orthomyxoviruses (influenza viruses) Influenza virus infections in humans: Clinical findings

37. Classic Flu-like Symptoms Fever Malaise (physical discomfort, mild sickness) Myalgia (muscular pain) Sore throat (inflammation of the fauces and pharynx) Nonproductive cough

38. Symptoms in children a) Higher fever b) GI symptoms-abdominal pain and vomiting c) Otitis media ( 中耳炎） d) Croup e) Myositis (muscle inflammation)

39. Reye’s syndrome An acute encephalopathy of children and adolescents High (10-40%) mortality rate A recognized complication of influenza B, A and herpesvirus varicellazoster infections Aspirin is to be avoided in children because of the association with Reye’s syndrome

40. Orthomyxoviruses (influenza viruses) Influenza virus infections in humans: Recovery

41. RECOVERY INTERFERON - SIDE EFFECTS INCLUDE: – FEVER, MYALGIA （肌痛）, FATIGUE （疲劳）, MALAISE （不适） CELL-MEDIATED IMMUNE RESPONSE TISSUE REPAIR – CAN TAKE SOME TIME

42. PROTECTION AGAINST RE-INFECTION IgG and IgA – IgG less efficient but lasts longer antibodies to both HA and NA important – antibody to HA more important (can neutralize)

43. Orthomyxoviruses (influenza viruses) Influenza virus infections in humans laboratory diagnosis

44. DIAGNOSIS ISOLATION – NOSE, THROAT SWAB – TISSUE CULTURE OR EGGS SEROLOGY – Hemagglutination inhibition, HI/ RAPID TESTS provisional - clinical picture + outbreak

47. Orthomyxoviruses (influenza viruses) Influenza virus infections in humans Prevention and treatment by drugs

48. VACCINE inactivated egg grown sub-unit vaccine for children reassortant live vaccine approved 2003 – for healthy persons (those not at risk for complications from influenza infection) ages 5-49 years

49. VACCINE ‘BEST GUESS’ OF MAIN ANTIGENIC TYPES – CURRENTLY type A - H1N1 type A - H3N2 type B each year choose which variant of each subtype is the best to use for optimal protection

51. Flu & Oseltamivirphosphate Oseltamivirphosphate

52. Emerging viral diseases: Avian influenza virus Orthomyxoviruses (influenza viruses) Crossing of species barrier ?

53. Influenza Hong Kong, 1997: death of 7.000 chicken in 3 farms  Crossing of species barrier of a new influenza strain (H5N1) from poultry to man  20 humans became infected. H5N1 One third die. No human to human transmission.  Killing of 1,6 millions poultry to prevent spreading among poultry  Vietnam, Thailand, China, Laos, Korea, Japan, Kambodscha, Indonesien, 2004: reemergence of H5N1  Suspected human to human transmission

54. H7N9

55. Paramyxoviridae Measles virus Mumps virus Parainfluenza virus Respiratory syncytial virus, RSV

56. -ssRNA

57. measles (rubeola) Koplik's spots on mucosal membranes Maculopapular rash (extends from face to extremities)

58. Sub-acute Sclerosing Panecephalitis (SSPE) Very rarely (7 in 1,000,000 cases) 1-10 years after initial infection progressive, fatal disease defective forms of the virus in the brain

59. British "to mump" - to grimace or grin, MUMPS VIRUS & Mumps from the appearance of the patient as a result of parotid gland swelling.

60. parainfluenza virus & flu-like symptom

61. RESPIRATORY SYNCYTIAL VIRUS Upper respiratory infection (‘bad cold’) in older children and adults Lower respiratory infection- Bronchiolitis and/or pneumonia may occur after the upper respiratory infection Severe infections occur in pre-term infants

62. CORONA VIRUSES COLDS & SARS

66. The masked palm civet （果子狸）

68. Others

69. ADENOVIRUS non-enveloped linear double-stranded (ds) DNA Icosahedral capsid, capsomeres hexons; at the vertices are 12 pentons, from which a fiber with a terminal knob projects. This complex is toxic to cells - causing rounding and death of cells through inhibition of protein synthesis.

70. Eye Epidemic Keratoconjunctivitis (EKC), acute follicular conjunctivitis, pharyngoconjunctival fever Respiratory system Common cold (rhinitis), pharyngitis (with or without fever), tonsillitis, bronchitis, pharyngoconjunctival fever, acute respiratory disease (LRI) Genitourinary Acute hemorrhagic cystitis Gastrointestinal Gastroenteritis.

71. RUBELLA (GERMAN MEASLES) VIRUS Togavirus +ssRNA Fetal damage live vaccine (attenuated strain)

72. RUBELLA Rash Congenital rubella 皮疹 先天性风疹

73. Summary 1. Understand the structure of influenza virus 2. Know the classification and nomenclature of influenza viruses 3. Master the function of hemagglutinin and neuraminidase ; master the relationship between antigenic drift/shift with flu epidemic or pandemic. 4. Know the procedure for influenza virua replication. 5. Understand the pathogenesis, pathology, clinical findings, laboratory diagnosis, epidemiology, prevention and control of influenza virus infection.

74. 1. 2. 3. Self control questions The principal reservoir for the antigenic shift variants of influenza virus appears to be: (A) People in isolated communities such as the Arctic (B) Animals, specifically pigs, horses, and fowl (C) Soil, especially in the tropics (D) Sewage Each of the following statements regarding influenza virus is correct EXCEPT: (A) Influenza A virus causes more epidemics and more serious disease than influenza B and C viruses do (B) Influenza viruses cannot be grown in cell cultures; hence, the diagnosis can only be made serologically (C) Influenza A virus undergoes major antigenic changes in its hemagglutinin (antigenic shift), which allow the virus to evade existing immunity (D) Influenza viruses are transmitted primarily by aerosol and primarily affects the lower respiratory tract Each of the following statements concerning the antigenicity of influenza A virus is correct EXCEPT (A) Antigenic shifts, which represent major changes in antigenicity, occur infrequently and are due to the recombination (reassortment) of segments of the viral genome (B) Antigenic shifts affect both the hemagglutinin and the neuraminidase (C) The worldwide epidemics causes by influenza A virus are due to antigenic shifts (D) The protein involved in antigenic drift is primarily the internal ribonucleoprotein

75. 4. 5. Self control questions Each of the following statements concerning influenza is correct EXCEPT: (A) Major epidemics of the disease are caused by influenza A viruses rather than influenza B and C viruses (B) Likely sources of new antigens for influenza A viruses are the viruses that cause influenza in animals (C) Major antigenic changes (shifts) of viral surface proteins are seen primarily in influenza A viruses rather than in influenza B and C viruses (D) The antigenic changes that occur with antigenic drift are due to reassortment of the multiple pieces of the influenza virus genome Biochemical analysis of a virus reveals the genome to be composed of eight unequally sized pieces of single- stranded RNA, each of which is complementary to viral mRNA in infected cells. Which one of the following statements is UNLIKELY to be correct? (A) Different proteins are encoded by each segment of the viral genome (B) The virus particle contains a virus-encoded enzyme that can copy the genome into its complement (C) Purified RNA extracted from the virus particle is infectious (D) The virus can undergo high-frequency recombination via reassortment of its RNA segments

76. Self control questions 6. Please explain the molecular reasons related to sporadic outbreaks, limited epidemics or pandemic of “flu”. 7. Where do where do “new” hemagglutinin & neuraminidase of influenza virus come from? 8. Please explain sub-acute sclerosing panencephalitis (SSPE) as an example of persistent slow infection for measles virus. 9. Term explanation: Antigenic drift and antigenic shift

77. Thank you so much!