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Rsv final


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Rsv final

  1. 1. Respiratory syncytial virus Baraa Alomar Supervised by: Prof. Fawza Mounem Damascus University Pharmacy faculty 1
  2. 2. The virus Taxonomy Viral structure Life cycle Immunity and Immunity evasion Pathogenesis diagnosis Treatment and prevention Contents 2
  3. 3. Introduction RSV primary cause of hospitalisation in the first year of life more than 120,000 infants are hospitalised annually with RSV infection 100% of children in the USA are infected with the virus by 2 to 3 years of age cause 3000 to 4000 deaths annually 3
  4. 4. Burden WHO indicate that (RSV) accounts worldwide for more than 60% of acute respiratory infections in children and more than 80% in infants younger than 1 year. RSV is the most frequent cause of pediatric bronchiolitis and pneumonia 4 ce=lnms&tbm=isch&sa=X&ved=0ahUKEwi5tqeR2M3MAhWmdpoKHacyAocQ_AUIBigB#imgrc=c8QHP9Zay6toCM%3A
  5. 5. 1st step RSV was noted in 1840s 2nd step First isolation in 1956 “chimpanzee coryza agent” 3rd step: Robert Chanock in 1963 characterized the virus and named it RSV History 5
  6. 6. Taxonomy Genome (-) ssRNA Virus Family Paramyxoviridae Subfamily Pneumovirinae Order mononegavirales. syncytia surface protein Why RSV??!HRSV RSV A subtype (11) (ON1, GA1–GA7, SAA1, NA1, and NA2) RSV B subtype (17) (GB1–GB4, SAB1– SAB3, and BA1– BA10) Nucleotide sequences Envelope glycoprotein 6
  7. 7. RSV 7
  8. 8. The viral genome (-)ssRNA molecule of 15,222 nucleotides linear order separated by short variable intergenic sequences, with the exception of the last two genes (M2 and L) that overlap by 68 nucleotides Each HRSV gene is framed between a gene- start (GS) sequence (3’-CCCCGUUUA(U/C), which is conserved in all genes except in the L gene which is slightly different 8
  9. 9. The Viral proteins NS 1, NS2 : Nonstructural proteins inhibit the induction of alpha/beta interferon Nucleoprotein: (N) It is bound tightly to the genome, forming the helical nucleocapsid Phosphoprotein: (P) protein Co-factor of the viral RNA polymerase (L) 22k (or M2-1) protein: Transcription processivity factor The RNA-dependent RNA polymerase (L): Polymerizing. Capping. Methylation. Polyadenylation. M2-2 protein: decreases transcription Induces replication SH protein: Inhibit apoptosis Fusion protein: (F protein) once the virus particle is bound to the host cell, exposure of the fusion peptide leads to its insertion in the cell membrane G protein: Transmembrane (Gm) is incorporated into virions transmembrane anchor domain. (M) protein: the M protein coordinates the association of the nucleocapsid complex with the cellular membrane 9
  10. 10. 00466-HTML-r2/image_m/fimmu-05-00466-g001.jpg RSV 10
  11. 11. replication of the viral genome involves the synthesis of a complementary antigenome (cRNA) Basics their genomes are tightly associated with the nucleoprotein (N) to form RNase-resistant nucleocapsids, transcription proceeds in a sequential and polar manner from the 3’-end of the viral RNA (vRNA) linear, negative-sense, single-stranded RNA molecule as the genome entry of the viral nucleocapsids into the host cells involves membrane fusion the virus particles are surrounded by a lipid bilayer in which the viral glycoproteins are inserted, 11
  12. 12. RSV life cycle old/rachel-fearns-phd/ Fusion assembly budding 12
  13. 13. RSV life cycle Fusion assembly budding 1st step (fusion) • binds to a specific cell surface component, such as CD46. • Attachment by G protein and fusion by F protein. 13
  14. 14. RSV life cycle Fusion assembly budd 2nd step (transcription) • is triggered once the viral nucleocapsid ha entered into the cell cytoplasm. • requires the coordinated action of at least four viral proteins: N, P, L and 22k. 14
  15. 15. RSV life cycle Fusion assembly budd 3rd step (replication) • Replication requires N, P and L proteins but not 22k. • require the action of another protein, M2-2. 15
  16. 16. RSV life cycle Fusion assembly budding 4th /5th step (assembly)/budding • different HRSV gene products accumulate near the cell • membrane where they are assembled into progeny virus particles that are released16
  17. 17. Life cycle can take place in enucleated cells. HRSV infection influences the expression of certain nuclear genes 17
  18. 18. Immunology of RSV18
  19. 19. Cellular infection triggers the release of early inflammatory mediators, e.g., TNF and IFN-α/β NK cells and PMN are recruited in the first 3 days of infection. DC carry viral antigen to local lymph nodes and present it to CD4+ T cells 19 Immunology of RSV
  20. 20. • Serum of humans various antibodies. • Only antibodies against the F or G glycoprotein are effected. B Lymphocytes :
  21. 21. • The N, SH, F, M, M2, and NS2 proteins stimulate RSV-specific CD8+ CTLs. • NK cells appear in the first few days after RSV infection. T Lymphocytes
  22. 22. NK and CD8+ T cells lymphokines (interferon-GAMMA). Lymphokines antiviral activities Activating (macrophae neutrophils). Resolve infection Damage tissue
  23. 23. Antibodies (F + G ) Stimulation CD8+ CTLs NK cells appearance Lymphokine secretion Summary
  24. 24. Immunology of RSV24
  25. 25. Adaptive immune response Prior RSV infection does not confer complete protection against reinfection immunity to RSV infection in the lung may be more durable than that in the upper respiratory tract (URT) adaptive immune response 25
  26. 26. NS1 and NS2 have been shown to suppress the production INF- ᵞ Changes in the pulmonary microenvironment are maintained in a relatively anti-inflammatory state RSV immunity evasion 26
  27. 27. RSV immunity evasion CD8+ T cells are impaired in their cytolytic ability and capacity to produce IFN-γ DCs are poor inducers of T cell effector function, they significantly impair CD4+ T cell proliferation 27
  28. 28. Pathogenesis of RSV in Children 28
  29. 29. wheezing dyspnea tachypnea poor feeding Bronchiolitis of RSV symptoms 29
  30. 30. Collection of clinical samples Nasopharyngeal swabs Nasal washes (pediatric patients) Collection of sputum Collection bronchial alveolar lavage fluids 30
  31. 31. Nasal washes (pediatric patients) Nasopharyngeal swabs (elderly patients) Sputum samples have higher titers than nasal swabs and offer higher diagnostic yield Notes for collection of samples 31
  32. 32. Viral culture Type of cell human lung fibroblasts primary rhesus monkey kidney Time for CPE primary rhesus monkey kidney (the earliest) human fibroblast (the slowest) Time of incubation 5-7 days 32
  33. 33. syncytia CPE of RSV Panq8bMAhVIBI4KHRj2BaoQ_AUIBygC#imgrc=IyXwG7l54iCJZM%3A 33
  34. 34. Used to measure serum immunoglobulin G (IgG) The antigens are: purified RSV envelope glycoproteins , The fusion (F) protein, the attachment protein. EIA for RSV 34
  35. 35. RT-PCR Detect minute quantities of RSV Rapidly identify acute infections Primers are used to amplify conserved portions of the F and N genes of RSV RT- PCR for RSV 35
  36. 36. ` P1 (5′-ACA ACA GAT CTC AGC AAA T-3′) P2 (5′-CTA TTG CTT GAT TGT CAC C-3′) P3 (5′-CTA TTG CTT GAT TGT CTC C-3′) Primers of conserved sequences in RSV The PCR products was analyzed by electrophoresis in a 1.2% agarose gel. The separated fragments were stained by ethidium bromide and visualized under UV light. 36
  37. 37. Detecting by direct immunofluorescence assay (DFA) •It is rapid and sensitive methods. •Princible: Infected cells fluorescent RSV- antibody examined by microscopy 37
  38. 38. Detecting by direct immunofluorescence assay (DFA) 38
  39. 39. Sensitivity and specificity rce=lnms&tbm=isch&sa=X&ved=0ahUKEwibm6vM8cjMAhWEbhQKHY8pBp0Q_AUIBigB#imgrc=tJihHATRoPywqM%3A 39
  40. 40. removal of secretions administration of oxygen bronchodilators Corticosteroids Treatment (supportive care) 40
  41. 41. Treatment (Antiviral agents) Antiviral agents severe RSV disease high-risk patients. inhibition of the replicating virus initiated at the onset of the infection Ribavirin an analogue of the nucleic acid guanosine 41
  42. 42. RSV Vaccination PFP subunit vaccine (purified F protein) Immunization of the elderly with chronic cardiac or pulmonary disease cpts live attenuated vaccine (cold-passaged temperature-sensitive) Immunization of young infants combinations of different types of vaccines will be needed for certain populations42
  43. 43. 43
  44. 44. • How many gens in RSV genome? • What is the function of (F) protein? • Which antiviral is approved for treatment RSV bronchiolitis? 44 Massages to take home
  45. 45. • Name tow type of RSV cell culture. • What is the CPE of RSV? • Which is the most sensitive methods for detecting RSV infection? 45 Massages to take home
  46. 46. • Robert A. Dudas and Ruth A. Karron, Respiratory Syncytial Virus Vaccines, Elsevier, 1998 Jul; 11(3): 430–439. • Gonzalez IM1, Karron RA. Eichelberger M. Walsh EE, Delagarza VW.Bennett R. Chanock RM.Murphy BR. Clements-Mann ML. Falsey AR, Evaluation of the live attenuated cpts 248/404 RSV vaccine in combination with a subunit RSV vaccine (PFP-2) in healthy young and older adults, Elsevier Vaccine. 2000 Mar 6;18(17):1763-72. • Ogra PL, Respiratory syncytial virus: the virus, the disease and the immune response,Elsevier Paediatr Respir Rev. 2004;5 119-26. Reference 46
  47. 47. • Piedimonte.G, Perez.M, Respiratory Syncytial Virus Infection and Bronchiolitis, pediatrics in review. December 2014;35(12):1-18. • Peter J. M. Openshaw and John S. Tregoning, Immune Responses and Disease Enhancement during Respiratory Syncytial Virus Infection, clinical microbiology review; July 2005vol. 18 no. 3 541-555. • Jose´ A. Melero, Molecular Biology of Human Respiratory Syncytial Virus in (Respiratory Syncytial Virus),Cane.P,Elsevier;2007(14).1-43. • Taylor.G, Immunology of RSV in (Respiratory Syncytial Virus),Cane.P,Elsevier;2007(14).43-89. 47 Reference
  48. 48. • Stephen P. Brearey, Rosalind L. Smyth, Pathogenesis of RSV in Children in (Respiratory Syncytial Virus),Cane.P,Elsevier;2007(14).1-43. • Becker.Y, Respiratory syncytial virus (RSV) evades the human adaptive immune system by skewing the Th1/Th2 cytokine balance toward increased levels of Th2 cytokines and IgE, markers of allergy--a review,springer link, 2006 Oct;33(2):235-52 • (7/5/2016) • 5/5/2016). • (3/5/2015) 48 Reference