1. INSIGHTS INTO PATHOGENESIS OF INFLUENZA (A) VIRUS ROLE OF SUSPENSION & (MDCK) TISSUE CULTURE IN DIAGNOSIS, VACCINE PRODUCTION
Dr. Ammar Jusmani/ m.m.b.s. MSc Clinical Microbiology (University of London)

2. INTRODUCTION
Influenza (A) virus is notoriously known to be responsible for the (1918) –(H1N1) pandemic
(1957)- (H2N2) Pandemic (SHIFT)= (REASSORTMENT)
(1968)- (H3N2) Pandemic (SHIFT)= (REASSORTMENT)
(2009)-((H1N1) Pandemic (SHIFT)= (REASSORTMENT)
Initially thought to be caused by the fastidious microorganism ( H. influenza), however the virus was finally detected in the early 1930s

5. FACTS ABOUT INFLUENZA VIRUS
Low copying fidelity (RNA) VIRUS
Belongs to the Ortho-myxo-viridae family
Three main classes (A, B, &C), but recently another type has been discovered (D)

6. STRUCTURE OUTER GLYCOPROTEINS : a) Hemaglutinin (HA)
b)Neuraminidase(NA)
Play balancing role in the pathogenesis, where (HA) initiates binding & entry into cell it intends to infect and (NA) releases newly assembled viruses (final stage in the life cycle of the virus)

7. Strc… contd…
(M1): Matrix protein (1)- Interacts with Rionucleoprotein (RNP)
(M2): matrix protein (2)- Ion channel for genome exit from virus during life cycle
(PB1): Polymerase( basic) protein (1)-Endonuclease function
(PB2): Polymerase (basic) protein(2)- Cap recognition
(PA): Polymerase (Acidic)
(NSP): Non-structural protein / (NEP): Nuclear Export Protein

9. VARIATIONS AMONG TYPES
(IAV) +(IBV): BOTH possess TWO major glycoproteins ( HA &NA)
(ICV) +(IDV)have ONLY ONE glycoprotein called Hemaglutinin Esterase Fusion Protein (HEF), which performs functions of both (HA & NA)
(HEF) in (IDV) is ACID STABLE and HEAT SATBLE : Important for pathogenesis.
Jeishi Yu, Busha Hika, Runxia Lu,…
The Hemaglutinin Esterase Effusion GlycoProtein is a primary determinant of the Exceptional Thermal &Acid Stability of Influenza(D) Virus
mSphere, American Association of Microbiology, July24, 2017.(doi: 10:1128/ msphere 0o254-17

10. INFECTIVITY REQUIREMENTS
(HA) requires –Cell Surface Receptors: either
(alpha 2,6) Sialylated HUMAN cell receptors or
(alpha 2,3) Sialylated AVIAN & EQUINE receptors .
SWINE : HAVE BOTH : ALLOWS (A, B, & C) ACCESS
(IAV)- INFECTS TIGERS, DOGS , SEALS, HORSES & HUMANS
(IDV)-INFECTS CATTLE, recent serological evidence of (IDV) IN HUMANS

11. INFECTIOUS STAGES
I) ENTRY INTO HOST CELL ( Nasal Mucosal Epithelium of the Upper Respiratory Tract)
OPTIMAL (HA) activation depends on suitable (PH) that matches viral type (HA)
II) ENTRY INTO CYTOPLASM: Low (PH) opens the (M2) ion channel that allows exit of the (VRNP) into cytoplasm.
III) ENTRY INTO NUCLEUS: (VRNP) imported via (NLS) –nucleus localization signals that respond to host import system to enter nucleus.
INSIDE NUCLEUS: (Transcription): first: Viral (RNA) strand converted to positive sense= template for production of (RNA) to be packaged into the new viral particles
Transcription: “Cap Snatching”: viral Endonuclease ( PB2) CLEAVES HOST (mRNA) to produce FRAGMENT

13. LIFE CYCLE
IV) EXPORT FROM NUCLEUS: The newly formed (vRNA) is exported from nucleus via (NEP) that binds to the (C-residue ) of the viral (M) protein = (CRM1).
V) ASSEMBLY & BUDDING:
(N.B.): (M2) ESSENTIAL TO VIRAL SHAPE

15. HIJACKING OF HOST CELL MACHINERY
1)Virus relocalizes host cell (RNA) components into nucleus of infected cells.
2)Virus prevents export of host cell (mRNA) from nucleus.

17. WHAT HAPPENS IN VIRAL CELL CULTURES ?
Peripheral Blood Mononuclear Cells(PBMCs) Isolated through ficoll paque method in
- Falcon tubes
-Centrifugation fro (30) minutes at (2250) rpm
-serum removed to reach the (PBMC) monolayer using a (1) ml. pipette
-Monolayer transferred to new (50) ml. falcon tubes filled with (RPMI), and centrifuged at (1,800) rpm for (10) mins-twice-

18. PREPARATION FO MACROPHAGES
Contents from preliminary steps poured into two separate flasks – one for the macrophages and another for the lymphocytes.
Macrophages are prepared by adding (GMCSF) to one of the flasks(incubated at (370C).
Lymphocytes prepared by adding IL-2 to other flask.(Prior to IL-2, PHA-phytohemaglutinin is added to PBMCs to maintain DNA synthesis for three days to stimulate cell surface receptor production.(incubated at 370 C.

19. VIABILITY OF LYMPHOCYTES
This is tested on the basis of the TOLUIDINE BLUE DYE TEST METHOD in a counting chamber (bauer manufacturers), using light microscopy: viable cells appear white and shiny
Dead cells appear darkly stained

20. INFECTION OF PBMCs
Infection done with viruses cultivated injected into the allantoic fluid and obtained after four days from egg grown human throat samples.
Presence of virus was confirmed using the Hemaglutination Inhibition Test (HAI).

21. VIRAL STRAIN (H3N2) : (IAV): A/ Sydney/ 05/ 97
-contains avian and human receptor analogue attachment sites.
-included in (2016/ 2017) vaccine program in certain countries in the world.
-cause of a pandemic (1968)
-no need to work in BSL-4 conditions as in the case with the more virulent form of H1N1)

22. DAYS OF (HAI) TESTING
FOLLOW UP DONE AT DAYS :(2,5,7,9,23,25,27,29) for lymphocytes and (2 weeks ) for macrophages
Macrophages and Lymphocytes exposed to same infective dose (EID: 1x107pfu & 1x108 pfu)
Macrophages and lymphocytes were then transferred to (MDCK) tissue culture.

23. (HAI) ASSAY TEST
(250 microL) Turkey blood cells( company frist elseviers 50% v/ v) added to (PBS) (50ml) .
Two fold dilutions of neat virus were transferred to microplates and incubated at (370 C) for twenty minutes.( PBMC supernatants were used).

24. Table (1): HA titers in lymphocytes (days 2 to 9)
Hemagglutination lab. Assay titers are all negative with both EID50 in the early stages of infection of peripheral blood lymphocytes.
HA titer
EID50 ( 1X107 pfu )
EID50 (1X 108 pfu)
Sample (day)
------
----- 2 5 7 9

25. EID50( 1X 107 pfu) EID50 (1X108 pfu) Sample (day) 1 : 2 1 : 4 23 25 27
Positive titer readings began from day 23 as shown in table (2) below
HA titer
EID50( 1X 107 pfu)
EID50 (1X108 pfu)
Sample (day)
1 : 2
1 : 4 23 25 27
-----
1 : 8 29

26. Table (3): HA titers in macrophages ( 2 weeks post- infection)
EID50
1 : 32
1 x 108 pfu
1 : 16
1x pfu

27. HA titers EID50 1X 107 pfu EID50 1X 108 pfu Sample (day) 1 : 64 23 25
Table (4) :HA titers in MDCK cell lines inoculated with lymphocyte supernatants
(days 23 to 29)
HA titers
EID50 1X 107 pfu
EID50 1X 108 pfu
Sample (day)
1 : 64 23 25 27
-----
1 : 128 29

28. Table (5 ):HA titers in MDCK lines inoculated with macrophages
EID50
1 : 256
1 X 108 pfu
1 : 128
1 X 107 pfu

29. MACROPHAGES
-The high titers in macrophages explained on the basis that macrophages have additional receptors that act as binding sites for influenza virus other than the classical sialylated (alpha 2,3 or 2,6 ) receptors .
-The additional receptors have recently been revealed and are called Macrophage Mannose Receptors (MMR) & (Lectin) receptors,
-They act as Co-receptors with the sialylated cell surface receptors.
-The high titers in Macrophage cell cultures do not necessarily indicate presence of viable virus, because replication in macrophages is abortive;
-macrophages release pro-inflammatory cytokines

30. LYMPHOCYTES
-Low titers explained on the basis of cell surface receptor transcription process that takes place in a gradual fashion and therefore is time related.
-Apoptosis: Influenza directs lymphocytes into a programmed cell death through (FAS) receptors.
-both (T & B) lymphoctes counts were found to be reduced in humans afflicted with influenza.

31. MDCK MDCK contains (HA) binding sites :
It is for this reason that MDCK has recently been proposed to be used for propagating influenza virus for the purpose of:
--manufacturing vaccines
-I.D. virus strains in lab. during surveillance
-confirmation of lab.diagnosis
-anti-influenza drug testing (neuraminidase inhibitors- Oseltamnivir)

32. SPECIMENS / TESTS TECHNICAL REASONS
SALIVA/DFA
PBMC/ Peripheral blood
-Paucity of epithelial cells
(viral particles reside inside (cells rather than saliva
-Sputum may contaminate saliva
--Container with saliva
necessitates the presence of (VTM)
-Risk of spread of Nosocomial infections
-DFA high quality clinical specimens& lab personnel
Obtained through simple blood sample - no risk of nosocomial infection
-(HAI) is all you need
-Test useful even if macrophages abort infection
-Incubation of PBMCs at 370

33. TESTS MOLECULAR REASONS
DFA
(HAI)
-testing for whole virus
Testing for (HA) molecule:
-linked to virulence
-linked to antigenicity
-linked to receptor binding
-linked to immune evasion by drift & shift mechanism

34. REFERENCES Influenza: Exposing the true killer
a1)Heather L. Van Epps
Influenza: Exposing the true killer
J Exp Med Apr 17; 203 (4) :803
doi: 10: 1084/ jem.2034ft
2)Tanis Dingle
Rapid Influenza Testing: Three Key Questions for Laboratorians
Bench Matters: September 2016
3)John Y. Takekawa, Nichola J. Hill, JosephP. Dudley
Rapid Diagnosisof Avian Influenza Virus in Wild Birds:
Use of portable Rrt-PCR and Freeze Dry Reagents in The Field
J. Vis. Exp 2011; (54) 2829
doi: ? 2829

35. REFERENCES 5)Cox NJ Global Epidemiology of Influenza: past and present
4)Sandra Okubo, Michael Cheung, Felice C. Adler-Shohet.
Comparing Indirect Fluorescent Antibody Assays to Rapid DiagnosticTests for The Detection of Influenza A (H1N1pdm09)
Medical Laboratory Observer September 1, 2012
5)Cox NJ
Global Epidemiology of Influenza: past and present
Annu Rev Med 2000

36. REFERENCES 6)Tasleem Sanji
Influenza A: Understanding The Viral Life Cycle
Yale J. Biol Med 2009 Dec; 82(4):
7)Pinto LH, Lamb RA
The M2 Proton Channels of Influenza A& B Viruses
J. Biol Chem. 2006; 281(14):
8)Marriette F. Ducate, Claire Pellletier, & Gilles Mayer
Influenza D Virus in Cattle, France( )
Emerging Infectious Diseases (CDC)
Volume 21, Number 2- February2015

37. REFERENCES 10)Peripheral Blood Mononuclear Cells:
9)Marriette F. Ducate, Claire Pellletier, & Gilles Mayer
Influenza D Virus in Cattle, France( )
Emerging Infectious Diseases (CDC)
Volume 21, Number 2- February2015
10)Peripheral Blood Mononuclear Cells:
Isolation, Freezing, Thawing and Culture
Protocol Vol. 1304
Methods in Molecular Biology pp. 53 – 61
Date 5 August 2014
Springer

38. REFERENCES
11)Jaqueline P. Upham, Danielle Pickett, Tatsuro Irimura, E Margot Anders, Patrick C Reading
Macrophage Receptors for Influenza Virus. Role of the Macrophage Galactose- type Lectin and Mannose Receptor in Viral Entry.
Journal of Virology 27 January 2010 vol 84 no – 3737
Doi: /JVI

39. REFERENCES 12)Se Pyeong Im, Tae Sung Jung
Invevariable lymphocyte receptors In the alternative adaptive immune response of hagfish
stigation of Developmental and Comparative Immunology
Science Directvol55, February 2016, pages 203 – 210
13)Joan E. Nichols, Jean A. Niles, and Norbert J. Roberts Jr.
Human Lymphocyte Apoptosis after Exposure to Influenza A Virus
Journal of Virology July 2001 vol. 75 no – 5929
Doi: /JVI

40. REFERENCES
14)Ding Yuan Oh, Ian G. Barr, Jenny A. Mosse, and Karen . Laurie
MDCK SIAT 1 CELLS show improved isolation Rates for Recent Human Influenza Viruses Compared to Conventional MDCK Cells
Journal of Clinical Microbiology July 2008 Vol. 46 no
Doi: / JCM – 08

41. 15)Kalvin KWTO, LuLu, Cyril CYYiP, Rosana WS Poon, Ami MY Fung
Additional Molecular Testing of Saliva Specimens improves the detection of respiratory viruses.
Emerging Microbes and Infections
(2017) 6 e49/doi: /emi
16)Tsuruoka, H, Xu, H, Kuroda, K.
Detection of influenza virus RNA In peripheral blood mononuclear cells of influenza patients. Internal Medicine, 1997; 50: