Co-infection of epithelial cells established from the upper and lower bovine respiratory tract with bovine respiratory syncytial virus and bacteria

1. PUTU EKA SUDARYATMA
Graduate School of Medicine and Veterinary Medicine;
Laboratory of Veterinary Microbiology,
University of Miyazaki
Co-infection of epithelial cells established from
the upper and lower bovine respiratory tract with
bovine respiratory syncytial virus and bacteria
Dissertation defense, 2020/07/28

2. https://doi.org/10.1016/j.vetmic.2019.06.010
Received 25 April 2019; Received in revised form 6 June 2019; Accepted 12 June 2019

3. USA
• US $500 million per year
• 70-80% morbidity (Hilton et. al., 2014)
JAPAN
•41% cases in beef and dairy cattle
•(MAFF-Japan, 2016)
AUSTRALIA
•50% death calves with 70% severe clinical
sign cases (Sackett et. al., 2006)
IRAN
•50% respiratory pathogen infection in
dairy cattle (Shirvani et. al, 2011)
Economic losses
Bovine Respiratory Diseases Complex
(BRDC)

4. BRDC
Pathogens
Viruses Bacteria
Cattle
condition
Stress
Gershwin et al., 2016

5. • BRSV (Bovine respiratory syncytial virus)
• BCoV (Bovine coronavirus)
• BoHV-1 (Bovine herpesvirus-1)
• BPIV-3 (Bovine parainfluenza virus-3)
• BVDV (Bovine viral diarrhea virus)
Pasteurella multocida (PM)
Mannheimia haemolytica
Mycoplasma bovis
Histophilus somni
Gershwin et al., 2016
BacteriaViruses

6. Viruses and bacteria interaction in respiratory tract
Normal condition Virus infection
: Bacteria
: Viruses
: Bacteria

7. 50%
30%
20%
Asymptomatic diseases
n= 10
8%
53%
21%
18%
Pneumonia history
n= 38
10%
34%
28%
28%
Respiratory diseases
n= 29
33%
13%
8%
17%
29%
Non-respiratory diseases
n= 24
(-) (+) single (bacteria/virus) (+) > 1 virus (+) > 1 bacteria (+) multi-patogens virus & bacteria
Existences of Bovine Respiratory Pathogens in Miyazaki’s cattle
(Nakamura, Thesis, 2018)

8. Multi-pathogens Virus & Bacteria
V & V
10%
B & B
40%
Virus
&
Bacteria
50%
BRSV + PM
17%
BVDV + PM
15%
BoHV + PM
15%
BVDV + MB 3%
Pattern of Bovine Respiratory Multi-pathogens
(Nakamura, Thesis, 2018)

9. 0
25
50
75
100
24 48 72
BRSV infection increased P. multocida adherences
to human epithelial cells
0
25
50
75
100
24 48 72
Bacteria/cell
hpi
*
*
A549
* *
HEp-2
P. multocida
Uninfected
BRSV-infected
(72-hpi)
(Sudaryatma et al., Vet. Microbiol., 2018)
BRSV-infected
Uninfected

10. ?
We still have a big question about
BRSV and P. multocida interaction in cattle..???

11. BRSV and P. multocida interaction in cattle
Bovine respiratory epithelial cells
field cases
in vitro
(human cells)
Objective

12. “Japanese black cattle”
(Takasaki Meat Inspection Centre, Miyazaki)
Trachea
(bTEC)
Bronchus
(bBEC)
Lung (bLEC)
Establishment of Bovine Respiratory Epithelial Cells
(BRECs)

13. Primary
BRECs
Purification of epithelial cells from primary BRECs
Different-trypsinization times Pre-plating
remove
by washing
2nd Trypsinized,
15 mins
1st Trypsinized,
5 mins
Plating,
2 h

14. Cytokeratin Vimentin NucleiContrast Overlay
EBTr
bTEC
bBEC
bLEC
Immunofluorescence staining of purified BRECs
Passages 5

15. bTEC bBEC bLEC
Characteristics of purified BRECs
0
10
20
30
40
50
60
1 2 3 4 5 6 7
No.ofcells(104)/ml
days of culture
bTEC
bBEC
bLECP5
P10
P10

16. BRECs:
1. Trachea (bTEC)
2. Bronchus (bBEC)
3. Lung (bLEC)
BRSV
infection
(MOI 1)
• Cytopathic effects
• Viral load
• Cytokine expression
BRECs infected with BRSV

17. Culture medium
0
2
4
6
8
10
1 3 6 12 24 72 120 168
ViralRNA(log10copies/ml)
bTEC
bBEC
bLEC
*
*
*
**
*
BRSV (MOI 1)
* p < 0.05
hpi
BRSV-Alexa 594
Intracellular (3 dpi)
bTEC bBEC bLEC
CPE (7 dpi)
bTEC bBEC bLEC
Susceptibility of BRECs infected with BRSV

18. Cytokines mRNA expression of BRSV-infected BRECs
bIL-1 bIL-6 bTNF-
Foldchange
0
5
10
15
20
25
30
bTEC bBEC bLEC
0
5
10
15
20
25
30
bTEC bBEC bLEC
0
5
10
15
20
25
30
bTEC bBEC bLEC
Uninfected
BRSV-infected

19. Co-infection in BRECs
Uninfected PM
BRSV
(MOI 1, 3-dpi)
PM
(MOI 100, 1-hpi)
?BRECs:
1. Trachea (bTEC)
2. Bronchus (bBEC)
3. Lung (bLEC)

20. PM adherences to cells
BRSV RNA
2
3
4
5
6
7
0
20
40
60
80
100
uninfected
1dpi
2dpi
3dpi
2
3
4
5
6
7
0
20
40
60
80
100
uninfected
1dpi
2dpi
3dpi
2
3
4
5
6
7
0
20
40
60
80
100
uninfected
1dpi
2dpi
3dpi
Bacterial/cell
ViralRNA(log10)
bTEC bBEC bLEC
*
*
*
*
*
a, b : p < 0.05 (between uninfected cells)
* : p < 0.05 (BRSV vs. uninfected cells)
a
b b
P. multocida adherences to BRSV-infected BRECs

21. 0
10
20
30
40
50
bTEC bBEC bLEC
0
10
20
30
40
50
bTEC bBEC bLEC
Foldchange
(normalizedbybGAPDH)
0
10
20
30
40
50
bTEC bBEC bLEC
BRSV PM BRSV+PM
bIL-1 bIL-6 bTNF-
*
*
*
*
*
* *
*
*
Cytokines mRNA expression of co-infection in BRECs

22. BRSV
Z factor..???
Y factor..???
Pasteurella multocida
Summary of results

23. What factor related with P. multocida
adherences in upper and lower respiratory
tract after infected with BRSV..???
?

24. HRSV infection increased S. pneumoniae
adherence, by upregulated:
• ICAM-1
• CEACAM-1
• PAF-R
(Avadhanula et al., 2006; Yokota et al, 2012)
Influenza A virus (H1N1) infection increased
S. pneumoniae adherence, by upregulated:
• PAF-R
(Avadhanula et al., 2006)
Human coronavirus (HCoV-NL63) infection
increased S. pneumoniae adherence, by
upregulated:
• PAF-R
(Golda et al., 2011)
Human rhinovirus (RV-16) infection
increased S. pneumoniae adherence, by
upregulated:
• PAF-R
• CEACAM-1
• Fibrobectin
(Ishizuka et al., 2011; Wang et al., 2009)
Human metapneumovirus infection
increased S. pneumoniae adherence, by
upregulated:
• PAF-R
• CEACAM
(Lai et al., 2016)
Factor related to bacteria adherent in human respiratory diseases

25. 16 29
23
2
1
0
0
Downregulated
bTEC bBEC
bLEC
ICAM-1
46 84
11
12
1
0
1
Upregulated
PAF-R
bTEC bBEC
bLEC
Proteomic analysis of BRECs infected with BRSV
(Sudaryatma et al., Front. Microbiol. 2020)(Sudaryatma et al., Vet. Microbiol. 2020)

26. Upper Respiratory Tract
• BRSV infection decreased ICAM-1 in upper respiratory tract
• P. multocida adherence to upper respiratory tract is ICAM-1 dependent
GAPDH
ICAM-1
BRSV
0.1 1 1
Live UV−
(MOI) 0 Control ICAM-1
Mock
P. multocida (MOI 100)
Nuclei
siRNA
(Sudaryatma et al., Vet. Microbiol. 2020)
bTEC bTEC

27. Lower Respiratory Tract
• BRSV infection induces PAF-R expression
• BRSV-induced PAF-R expression enhances P. multocida adherence
• PAF-R is binding molecule for P. multocida
0
20
40
60
80
100
cont
-
sicont
si1P
si2P
Bacteria/cell * *
Control
Mock
Control
PAFR1
PAFR2
BRSV (MOI 1)
siRNA
PAFR
GAPDH
0
20
40
60
80
100
0 0 1 5 25
*
*
Anti-PAFR (μg/ml) 0 0 1 5 25
BRSV − ＋ ＋ ＋ ＋
PM ＋ ＋ ＋ ＋ ＋
Bacteria/cell
(Sudaryatma et al., Front. Microbiol. 2020)
bLECbLEC
pCEP4-bPAFR (0 µg) pCEP4-bPAFR (1 µg)
pCEP4-bPAFR(APC)
P. multocida (FITC)
HEK293T

28. Schematic model of severe pneumonia
by co-infection BRSV and P. multocida
“Gateway”
BRSV
PAF-R
ICAM-1
Pasteurella multocida

29. Differences in susceptibility of BRECs to BRDC-
related pathogens shed new light upon the
mechanism of severe pneumonia in cattle
Conclusion

30. Summary of PhD course research
(Sudaryatma et al., 2018)
BRSV increased PM adherence
in human epithelial cells.
Bovine respiratory cells?
(Sudaryatma et al., 2019)
Differences susceptibility of BRECs infected with
BRSV to response of PM adherence in bovine
respiratory cells.
What factor related to differences susceptibility BRSV
infection in upper and lower respiratory tract
(Sudaryatma et al., 2020)
(Sudaryatma et al., 2020)

31. Acknowledgments
PhD supervisors:
Tamaki Okabayashi
Yoshitaka Goto
Ryuichiro Atarashi
Members of:
Lab. Veterinary Microbiology, UoM
CADIC and staff
Bambang Foundations
PPI Miyazaki
Special Thanks:
MEXT, Japan
Bambang Pontjo P.
Hirohisa Mekata
Meiko Kubo
Akatsuki Saito
Naoaki Misawa

32. THANK YOU for supporting me in everything

33. THANK YOU

37. Primary BRECs (24 h seeding)
Trachea Bronchus Lung

38. Different trypsinization time
Trachea Bronchus Lung

40. BRSV-infected BRECs
BRSV-Alexa 594
Intracellular
(3 dpi)
bTEC
bBEC
bLEC
CPE
(7 dpi)
Culture medium
0
2
4
6
8
10
1 3 6 12 24 72 120 168
ViralRNA(log10copies/ml)
bTEC
bBEC
bLEC
*
*
*
**
*
BRSV (MOI 1)
* p < 0.05
hpi

41. (Chang Y-J et al., Mol., 2004)
Signalling pathways ICAM-1 expression in epithelial cells
(Kolli et al., Pathogens, 2013)

42. Garcia C.C., et al., 2020, The development of anti-inflammatory drugs for infectious diseases. Discov Med.
Role of PAFR regulate lung injury

43. Upper and Lower respiratory tract of cattle
Upper Respiratory (bTEC)Lower Respiratory (bLEC)

44. Surface protein change from BRECs infected with BRSV
0
0.5
1
1.5
- + - + - +
-foldchange
*
BRSV
bTEC bBEC bLEC
BRSV − ＋ − ＋ − ＋
GAPDH
ICAM-1
bTEC bBEC bLEC
ICAM-1
BRSV − ＋ − ＋ − ＋
GAPDH
PAF-R
bTEC bBEC bLEC
PAF-R
0
1
2
3
4
- + - + - +
-foldchange
*
*
BRSV
bTEC bBEC bLEC
(Sudaryatma et al., Front. Microbiol. 2020)(Sudaryatma et al., Vet. Microbiol. 2020)

45. Upper Respiratory Tract
• BRSV infection decreased ICAM-1 in upper respiratory tract
• P. multocida adherence to upper respiratory tract is ICAM-1 dependent
GAPDH
ICAM-1
BRSV
0.1 1 1
Live UV−
(MOI) 0
Control ICAM-1
Mock
P. multocida (MOI 100)
Nuclei
siRNA
bTEC
0
20
40
60
0 1 5 25
Bacteria/cell
*
*
ICAM1-Ab
(μg/ml)
PM (MOI 100)
bTECbTEC
(Sudaryatma et al., Vet. Microbiol. 2020)
ICAM-1 is BRSV-dependent PM decreased in Ab-cover PM decreased in ICAM-1 silencing

46. Lower Respiratory Tract
• BRSV infection induces PAF-R expression
• BRSV-induced PAF-R expression enhances P. multocida adherence
• PAF-R is binding molecule for P. multocida
GAPDH
PAFR
0
20
40
60
80
100
cont
-
sicont
si1P
si2P
Bacteria/cell
* *
Control
Mock
Control
PAFR1
PAFR2
BRSV (MOI 1)
siRNA
PAFR
GAPDH
0
20
40
60
80
100
0 0 1 5 25
*
*
Anti-PAFR (μg/ml) 0 0 1 5 25
BRSV − ＋ ＋ ＋ ＋
PM ＋ ＋ ＋ ＋ ＋
Bacteria/cell
(Sudaryatma et al., Front. Microbiol. 2020)
bLECbLECbLEC
MOI
BRSV − ＋ ＋ −
UV-BRSV − − − ＋
0
1
2
3
4
0 0.1 1 1
PAFR/GAPDH
*
*

47. Lower Respiratory Tract
• PAF-R is binding molecule for P. multocida
pCEP4-bPAFR(APC)
P. multocida (FITC)
Ginkgolide-B 0.9 µM Ginkgolide-B 28.8 µM
21.8%64.8%
Over-expression of PAF-R
&
PAF-R antagonist treatment
pCEP4-bPAFR (0 µg) pCEP4-bPAFR (1 µg)
pCEP4-bPAFR(APC)
P. multocida (FITC)
HEK293T
Over-expression of PAF-R
pCEP4-bPAFR (1 µg)
73.1%10.8%
(Sudaryatma et al., Front. Microbiol. 2020)

48. Stimulated cytokine
(bIL-1𝛃↑, b IL-6↑, b TNF𝛂↑)
Normal condition
BRSV infection
Trachea Bronchus Lung