A PRESENTATION DESCRIPS RESPERATORY INFECTIONS CAUSED BY RSV AND PATHOGENESIS , DIAGNOSIS , TREATMENT, VACCINATION,STRUCTRUE AND LIFE CYCLE OF THIS VIRUS

1. Respiratory syncytial
virus
Baraa Alomar
Supervised by: Prof. Fawza Mounem
Damascus University
Pharmacy faculty
1

2. The virus
Taxonomy
Viral structure
Life cycle
Immunity and Immunity evasion
Pathogenesis
diagnosis
Treatment and prevention
Contents
2

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. 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
https://www.google.com/search?q=Etiology+of+acute+respiratory+infections+in+children&biw=1366&bih=640&sour
ce=lnms&tbm=isch&sa=X&ved=0ahUKEwi5tqeR2M3MAhWmdpoKHacyAocQ_AUIBigB#imgrc=c8QHP9Zay6toCM%3A

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. 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. RSV
7
http://www.vtherm.com/rsv-bronchiolitis/

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. 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. http://www.frontiersin.org/files/Articles/112039/fimmu-05-
00466-HTML-r2/image_m/fimmu-05-00466-g001.jpg
RSV
10

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. RSV life cycle
http://www.bumc.bu.edu/microbiology/people/faculty-
old/rachel-fearns-phd/
Fusion
assembly
budding
12

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. 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. 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. 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. Life cycle can take place in
enucleated cells.
HRSV infection influences
the expression of certain
nuclear genes
17

18. Immunology of RSV18

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. • Serum of humans various antibodies.
• Only antibodies against the F or G glycoprotein
are effected.
B Lymphocytes :

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. NK and CD8+ T cells
lymphokines (interferon-GAMMA).
Lymphokines
antiviral
activities
Activating
(macrophae
neutrophils).
Resolve
infection
Damage
tissue

23. Antibodies (F + G )
Stimulation
CD8+ CTLs
NK cells appearance
Lymphokine secretion
Summary

24. Immunology of RSV24 http://cmr.asm.org/content/18/3/541/F2.large.jpg

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. 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. 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. Pathogenesis of RSV in Children
28

29. wheezing dyspnea tachypnea
poor feeding
Bronchiolitis of RSV symptoms
29

30. Collection of clinical samples
Nasopharyngeal swabs
Nasal washes (pediatric patients)
Collection of sputum
Collection bronchial alveolar lavage fluids
30

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. 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. syncytia
CPE of RSV
www.google.com/search?q=rsv+cpe&biw=1366&bih=640&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjM-
Panq8bMAhVIBI4KHRj2BaoQ_AUIBygC#imgrc=IyXwG7l54iCJZM%3A
33

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. 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. `
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. Detecting by direct immunofluorescence
assay (DFA)
•It is rapid and sensitive methods.
•Princible:
Infected
cells
fluorescent
RSV-
antibody
examined
by
microscopy
37

38. Detecting by direct immunofluorescence
assay (DFA)
38

39. Sensitivity and specificity
https://www.google.com/search?q=sensitivity+and+specificity+of+rsv+detection+methods&biw=1366&bih=640&sou
rce=lnms&tbm=isch&sa=X&ved=0ahUKEwibm6vM8cjMAhWEbhQKHY8pBp0Q_AUIBigB#imgrc=tJihHATRoPywqM%3A
39

40. removal of
secretions
administration
of oxygen
bronchodilators
Corticosteroids
Treatment (supportive care)
40

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. 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

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. • 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. • 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. • 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. • 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
• http://www.cdc.gov/flu/professionals/diagnosis/molecular-assays.htm
(7/5/2016)
• http://medical-dictionary.thefreedictionary.com/lymphokine
5/5/2016).
• http://www.dana.org/Media/GrantsDetails.aspx?id=38677 (3/5/2015)
48
Reference