Presentation on Zika

1. Presented by:
Dr. Mousumi Alam
Resident, phase-A, MD,
Department of Virology,
BSMMU.

2. Zika Virus:
 Family: Flaviviridae
 Genus: Flavivirus,
 Enveloped and icosahedral,
 has a nonsegmented, single-stranded, 10
kilobase positive-sense RNA genome.
the RNA genome encodes 7 nonstructural
proteins (NS1, NS2A, NS2B, NS3,
NS4A, NS4B & NS5) & 3 structural
proteins (PrM), Enveloped & capsid
protein.

3. Routes of Transmission:

6. Background:
• Wide-spread prevalence of the mosquito vector (Aedes spp.) & lack of approved vaccines and therapeutics
against Zika.
• This virus can be detected in human semen several months after infection.
• Pregnant women in non-endemic areas-at risk of acquiring ZIKV through sexual transmission from male
partners returning from endemic areas.
• Sertoli cells-supports spermatogenesis and maintenance of spermatogonial stem cells (SSCs) population that
eventually give rise to mature sperm.
• Leydig cells-produce and secrete the male sex hormone testosterone.

7. Materials and Methods:
• Cell culture:
• 3 Cell lines were used-
• Human sertoli cells
• Human leydig cells
• A549 cell line
• Virus strains used to infect the cells-
• African MR 766
• American Puerto Rican PR
Sertoli cell: more permissive
to ZikV infection.
Rest of the studies: performed
on this cell line.

8. Materials and Methods:
Sertoli cell
Measurement
of IFNβ, IFN
λ2 & OAS-2
Receptor
identification
(TIM & TAM)
Viral
persistence
Genetic
dysregulation
Role of FGF2
in viral
replication
NS1
measurement

9. Materials and Methods:
• Immunostaining and confocal
microscopy: to confirm ZikV infection of
Sertoli cells.
• Quantitative Real time PCR.
• ELISA
• FGF2
• NS1
• RNAseq Analysis

11. To detect ZikV infection of the sertoli cell:
3 cell lines
were taken
Sertoli cell
High level of
viral
replication
Leydig cell
Low level of
viral
replication
A 549 cell
line
High level of
viral
replication
Infected with 2 strains of ZikV
(MOI-5)
1. African MR766 (MR)
2. Amercian Puerto Rican (PR)
Evidenced by:
1. PFU
2. qRT-PCR

12. Fig: Sertoli cells support high levels of ZIKV replication. Primary human Sertoli and Leydig cells and the human A549 cell
line were infected with ZIKV strain MR766 (MR) or PRVABC59 (PR) (MOI = 5) for 24, 48, 72 and 96 hours. At each time
point, supernatants were harvested and viral titers were determined by plaque assay (A) and qRT-PCR (B).

13. To detect antiviral response of sertoli cell:
To assess the antiviral
response of Sertoli cell &
A549:
Detection of mRNA of IFNβ,
IFN-λ2 & OAS-2 by qRT-
PCR.
Induction of these genes:
lower in sertoli cell in
comparison to A549.

14. Fig: Cells were harvested at each time point and viral replication (B), Interferon-β (C), Interferon-λ2
(D) and OAS2 (E) mRNA levels were determined by qRT-PCR.

15. To identify receptor for ZikV:
• ZikV exploits TIM & TAM family receptors while entering into astrocytes, skin fibroblast
& endothelial cells.
• So, does ZikV use the same receptor in case of Sertoli cell?
• To find out the answer, Blocking antibody to these receptors were added to cell line and
infected again with both strains.
• Examples of TIM & TAM family receptors:
• TIM 1, 4
• Axl, Tyro-3, Mer etc.

16. • Approximately 90% of sertoli cell
have Axl receptors.
• Anti Axl antibody treated cells have
lower viral replication.
• Other receptors did not significantly
affect viral replication.

17. To further confirm the dependence of ZikV
infection on Axl:
Sertoli cell
Pre-treated with Axl inhibitors
R428 and infected with both
strains
Significantly inhibited ZikV
replication in dose dependant
manner
Pre treated with PS/PE binding
compound Duramycin and
infected with both strains
Significantly inhibited ZikV
replication in dose dependant
manner

18. Duramycin treated cell infected with MR & PR
Axl Inhibitors R428 treated cell infected with MR &
PR

19. To observe viral persistence:
• Sertoli cells were infected with ZIKV
(MOI = 0.5) and then monitored for
viral replication in culture for up to 6-
weeks.
• At 6-weeks post-infection, ~15% of the
Sertoli cells exhibited detectable ZIKV
antigen.

20. Sertoli cell after infection:
• Transcriptome profiles in mock- and ZIKV-
infected (MOI = 5) cells at 48 h post-infection
using RNAseq analysis.
• 57% variance in ZikV infected cell after 48h
post infection.
• 23% variance in ZikV infected cell after
6weeks post infection.
• So, Genetic dysregulations were found in
ZikV infected cells. After 48 hours post infection After 6 weeks post infection

21. Genetic dysregulations after ZikV infection:
At 48 h post-infection
• 9,209 transcripts: differentially expressed.
Among these,
• 5,828 transcripts: upregulated and
• 3,382 Transcripts: downregulated.
• most highly induced mRNA transcripts: FGF2
At 6-weeks post-infection
• Only 42 transcripts: deregulated
• 40: upregulated
• 2: downregulated .

22. To assess the role of FGF2 in ZikV replication:
• One of the upregulated transcripts (after 48h and 6wks post infection)
was: Fibroblast Growth Factor 2. (according to RNAseq analysis)
• ELISA was done to confirm increased level of FGF2: Depending upon the
time point and strain of ZIKV used for infection, there was up to 65-fold
more FGF2 secreted from ZIKV-infected Sertoli cells.

23. Supernatants from Sertoli cells mock-treated or infected with ZIKV MR766 (MR) or PRVABC59
(PR) strains (MOI = 5) were subjected to ELISA to determine levels of FGF2 protein during acute
(A) and persistent (B) infection.
(A) (B)

24. To investigate the potential role of FGF2 in
ZikV infection:
Seroli cell Pre-
treated
Anti-FGF2
virus replication was
reduced in a dose-
dependent manner
(by up to 50%)
BGJ398
ZIKV replication
was reduced by up
to 80%
Azithromycin
viral replication was
reduced by over
75%
rFGF2
modest but
significant increase
in ZIKV replication

25. Figure: FGF-2 secretion from Sertoli cells supports ZIKV infection and persistence. (A–D) Sertoli cells were treated for 16-
hours with anti-FGF2 antibody (A), recombinant human FGF2 (B), FGFR inhibitor BGJ398 (C) or Azithromycin (D) and then
infected with ZIKV MR766 (MR) or PRVABC59 (PR) (MOI = 0.5). Cells were harvested 48-hours post-infection and viral
replication was determined by qRT-PCR analyses of total RNA extracted from cells.

26. Detection of ZikV by NS1:
• Robust secretion of NS1 from zikv-
infected sertoli cells with peak levels
reaching ~ 350 ng/ml between 48–72 h
post-infection.
• Secreted NS1 can be used as potential
immunodiagnostics marker for zikv
infection in semen

27. Discussion:
• ZIKV is unique among mosquito-transmitted flaviviruses in its ability to spread through sexual transmission
• The present study focused on the susceptibility and permissiveness of two major cell types (Sertoli cells and
Leydig cells) supporting spermatogenesis in human testes.
• Sertoli cells support high levels of virus replication & shedding for prolonged periods of time. Human Leydig
cells poorly supported virus.
• ZIKV infection of Sertoli cells was dependent on the cell surface receptor Axl.
• RNASeq analysis revealed that acute infection of Sertoli cells with ZIKV results in massive dysregulation of
host transcripts (>9,000).
• This experiments revealed a pro-viral role for FGF2 in ZIKV replication and persistence.

28. Recommendations:
• Because FGFR signaling inhibitors and azithromycin severely hampered viral replication, it may
be worth further exploring their utility as therapeutic options to block sexual transmission of ZIKV.
• Finally, it shows that infected Sertoli cells robustly secrete NS1 protein which could potentially be
exploited as an immunodiagnostic marker for rapid screening of semen from suspected cases of
ZIKV infection in male patients.