intrinsic pathways in a time and dose dependent manner

Cell and virus

Vero cell line is maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal

bovine serum and penicillin/streptomycin at 37°C incubator with 5% CO2.

Schmallenberg virus is kindly provided by Dr. Wim H. M. van der Poel (Wageningen

University in the Netherlands). Vero cells were infected with 0.1 and 0.01 MOI of SBV and

excess virus not adsorbed was removed after 1 hr of incubation. Cell viability examined

with trypan blue (15250061, Thermo Fisher) exclusion method.

Plaque titration

SBV titration is performed on Vero cells as described previously with some modifications. Briefly, Vero cell line was inoculated in

12-well cell culture plate and after 18 hr cells were infected with SBV dilutions. After 3

hr of incubation of virus-infected cells in 37°C incubator with 5% CO2 virus

dilutions, inocula were removed and 1:1 mixture of 2× DMEM and 2% carboxymethyl cellulose

(CMC) were dispersed to all wells. After incubation for 6 days cells were fixed with 10%

neutral buffered formalin solution and stained with crystal violet. Plaques were counted

and stock virus titration was calculated as PFU/ml.

DNA fragmentation assay

Cells infected with 0.1 and 0.01 MOI of SBV were collected on 0, 2, 6, 12, 18, 24, 36, 48

hr Staurosporine (35385.02, Serva, Heidelberg, Germany) was used as positive control for

apoptosis induction with final concentration of 1 µM/ml.

Apoptotic DNA ladder kit (K170, Biovision, California, U.S.A.) was used for detection of

DNA fragmentation as following the instructions of manufacturer’s. Briefly, approximately

2 × 106 cells were collected and cell pellets were suspended with extraction

buffer followed by centrifugation at 1,600 × g. RNase and proteinase were added to

supernatants of each samples and incubated at 50°C for 30 min. After incubation samples

were incubated at −20°C for 10 min after ammonium acetate and isopropanol addition.

Samples were centrifuged at 16,000 × g for 10 min for DNA precipitation after incubation.

DNA pellet were washed with 70% ethanol and centrifugation was repeated at 16,000 × g for

10 min. Ethanol was removed and tubes were incubated at room temperature for 15 min for

evaporation of ethanol residues. DNA samples were suspended in suspension buffer and

loaded on 2% agarose gel stained with ethidium bromide and visualized under UV light.

Flow cytometry analysis

Cells infected with 0.1 and 0.01 MOI of SBV were harvested on 0, 2, 6, 12, 18, 24, 36, 48

hr and were stained with Annexin V-FITC and PI with using Annexin V-FITC Apoptosis Kit

(K101, Biovision, CA, U.S.A.) for detection of apoptosis in flow cytometry. Briefly, cell

pellets were suspended with 1× binding buffer. Cell groups were determined as unstained,

FITC, PI and FITC+PI stained. After cells were stained with related dye, samples were

incubated in room temperature for 5 min. Flow cytometry analyses were maintained with BD

FACSAria II (Franklin Lakes, NJ, U.S.A.) after calibration with beads. Data analysis of

samples was carried out with BD FACSDIVA software.

Caspase assays

Caspase-3, caspase-8 and caspase-9 activities of cells infected with 0.1 and 0.01 MOI of

SBV were determined in Vero cells harvested on 0, 2, 6, 12, 18, 24, 36, 48 hr pi.

Caspase-3 (K106, Biovision), caspase-8 (K113, Biovision) and caspase-9 (K119, Biovision)

colorimetric assay kits were used for determination of caspase activations. After

harvesting, cell pellets were suspended in 50 µl of cell lysis buffer

followed by incubation on ice for 10 min. Samples were centrifuged at 10,000 × g for 1 min

and cytosolic extracts were transferred to fresh tubes. Protein concentration of samples

was determined and 250 µg of protein from each sample were dilute in

total volume of 50 µl of cell lysis buffer in a 96-well microplate. Into

each well 50 µl of 2× reaction buffer with 10 mM of dithiothreitol (DTT)

was added. DEVD-pNA, IETD-pNA and

LEHD-pNA substrates were added with final concentration of 200

µM to each sample for determination of caspase-3, caspase-8 and

caspase-9 activation, respectively. Plates were incubated at 37°C for 2 hr and read at 405

nm using a microplate reader.

RNA isolation and reverse transcription

RNA samples were isolated from cells with using High Pure RNA isolation kit (11 828 665

001, Roche, Mannheim, Germany). Briefly, cells pelleted and re-suspended in 200

µl PBS thereafter 400 µl of lysis/binding buffer added

to each sample and vortexed. Samples were transferred to filter tubes and centrifuged at

8,000 × g for 15 sec. Ninety µl DNase incubation buffer and 10

µl DNase I were mixed per sample and transferred to filter tubes

followed by incubation at room temperature for 15 min. Five hundred µl of

wash buffer I was added to tubes and centrifuged at 8,000 × g for 15 sec. After

centrifugation, 500 µl of wash buffer II was added to tubes and

centrifuged again at 8,000 × g for 15 sec. Two hundred µl of wash buffer

II was added to tubes and centrifuged at 13,000 × g for 2 min. Filter tubes were

transferred into microcentrifuge tubes and 100 µl of elution buffer were

added to upper reservoir of filter tubes. After centrifugation at 8,000 × g for 1 min,

eluted RNA samples were stored at −80°C until used for reverse transcription. Reverse

transcription of cDNAs were performed with Transcriptor First Strand cDNA synthesis kit

(04 897 030 001, Roche, Mannheim, Germany) using anchored-oligo (dT)18 and

random hexamer primers. Briefly, total RNA, anchored-oligo (dT)18 and random

hexamer primers were mixed in total volume of 13 µl and heated to 65°C

for 10 min in thermal cycler. Thereafter tubes were immediately cooled on ice. Mix of 4

µl transcriptor reverse transcriptase reaction buffer, 0.5

µl protector RNase inhibitor, 2 µl dNTP mix, 0.5

µl transcriptor reverse transcriptase were prepared for each samples

and dispersed to tubes in final volume of 20 µl. Reverse transcription

reaction was performed in following condition, 25°C for 10 min followed by 55°C 30

min.

PCR and real-time PCR

Vero cells infected with SBV were confirmed by PCR using two different primers sets for

amplification of S segment of SBV after reverse transcription (Table S1). Real-time PCR

was used to analyze expression levels of anti-apoptotic genes Bcl-2 and

Bcl-XL and pro-apoptotic genes Bak,

Bax and Puma. GAPDH and Beta actin were selected as

reference genes. Primer sets for these apoptosis-related genes were designed (Table S2)

and real-time PCR optimization were performed for all sets. cDNAs were subjected to

real-time PCR with SYBR Green I master mix (04707516001, Roche, Mannheim, Germany) in

total volume of 20 µl for each reactions and 95°C 10 sec, 60°C 10 sec,

72°C 10 sec profile was used. Each sample was studied in duplicate. Analyses were

performed with LightCycler 96 instrument (Roche, Mannheim, Germany). Normalized gene

expression levels of apoptosis-related genes were calculated with 2-(Cq gene of

interest −Cq reference gene) infected sample -Cq gene of interest −Cq reference gene)

noninfected sample formulation [22, 26] with distinct normalization both with GAPDH and

Beta actin.

Statistical analyses

One-way ANOVA and independent t-test were used for statistical analyses

in IBM SPSS Statistics 21 software (Chicago, IL, U.S.A.). Results were considered as

statistically significant at P<0.01. Graphics were created with

GraphPad Prism software (San Diego, CA, U.S.A.).

Infection of Vero cells with SBV

Vero cells were infected with SBV on Vero cells and were confirmed with RT-PCR and plaque

titration that was also carried out to determine titer of virus stocks (Figs. S1, S2,

Table S1). Vero cells infected with 0.1 and 0.01 MOI of SBV were visualized under inverted

microscope (DM IL LED, Leica, Wetzlar, Germany) and cells were controlled daily for

cytopathic effect (cpe). Cells infected with 0.1 MOI of SBV showed cpe first in 18 hr

post-infection (pi). In infection of 0.01 MOI of SBV first cpe were seen in 24 hr pi and

cpe of cells were much less in comparison to those cells infected with 0.1 MOI of SBV.

Schmallenberg virus causes DNA fragmentation in Vero cell line

To detect whether DNA fragmentation is induced in SBV infection, cells were collected on

0, 2, 6, 12, 18, 24, 36, 48 hr post-infection and DNA samples were isolated. DNA

fragmentation assay of 0.1 and 0.01 MOI of SBV infected cells were visualized on 2%

agarose gel. DNA fragmentation of SBV infected Vero cells was dependent on infection time

and dose of virus, as DNA fragmentation of 0.1 MOI of SBV infected cells initiated on 18

hr pi while 0.01 MOI of SBV infected cells showed first DNA fragmentation on 24 hr pi.

Positive control of apoptosis (Staurosporine) was positive in DNA fragmentation and mock

infected control cells showed no DNA fragmentation (Fig. 1).

Phosphatidylserine exposure on Schmallenberg virus infected Vero cell line

To address if SBV infected cells culminate in phosphatidylserine translocation, cells

stained with Annexin V-FITC/PI were analyzed in flow cytometry. The highest ratio of

phosphatidylserine exposure of Vero cells was detected in 48 hr pi in 0.1 MOI of SBV

infection with total rate of 37.84% (Fig. 2A), whereas in 0.01 MOI infected 48 hr pi cells showed 22.7% apoptotic cells in total

(Fig. 2B).

Schmallenberg virus activates caspase-3, caspase-8 and caspase-9

To determine whether the infected cells show significantly more caspase activation than

the mock infected in the different time and dose, effects of SBV infection on caspase-3,

-8 and-9 activation in Vero cells were measured by using colorimetric kits. According to

caspase activity assays, caspase-3, -8 and -9 activations in SBV infected Vero cell were

detected. All caspase activation data sets were shown as the mean ± standard error of the

mean (SEM) versus the mock infected group for both 0.1 and 0.01 MOIs (Fig. 3).

Significant differences are determined for the infection time points versus the mock

infected cells and between dose groups. According to results, caspase-3 activation was

significantly different at 36 hr pi and 48 hr pi in both of 0.1 MOI and 0.01 MOI infected

cells compared to the mock infected group (P<0.01) (Fig. 3A). For caspase-3, there is statistically

significance between 0.1 MOI and 0.01 MOI groups of cells at 2, 6 and 12 hr pi

(P<0.01) (Fig. 3A).

Results indicate that SBV induce apoptosis in Vero cells with activation of caspase-3 in

both viral doses.

In activation of caspase-8 there are significant differences determined between mock

infected group and 2, 6, 36 and 48 hr pi cells in 0.1 MOI (P<0.01).

Caspase-8 was activated significantly at 0.01 MOI of 12, 18, 24, 36 and 48 hr pi cells

versus the mock infection cells (P<0.01). Caspase-8 is slightly

activated in SBV infected cells but there is statistically significance between all doses

except for those of 36 hr pi (P<0.01). Unexpectedly, proportions of

activated caspase-8 at 2, 6, 12, 18 and 24 hr pi of 0.01 MOI are higher than 0.1 MOI

groups (Fig. 3B). Caspase-8 activation in SBV

infected cells indicates that extrinsic pathway is triggered by SBV.

Activation of caspase-9 indicated moderate but significant increases at 0.01 MOI and

interestingly, stayed almost unchanged during the time. At 0.01 MOI in 2, 12, 18, 24, 48

hr pi cells there was statistical significance compared to mock infection

(P<0.01) (Fig. 3C).

Statistically significance is determined at 0.1 MOI for 2, 6, 18, 36 and 48 hr pi groups

in comparison to mock infected group (P<0.01). Infected cells with 0.1

MOI of SBV has higher proportion of activated caspase-9, supporting SBV induce intrinsic

cascade (Fig. 3C).

Gene expression analyses of SBV infected Vero cells

After the induction of apoptosis in SBV infected Vero cells was confirmed by DNA

fragmentation, flow cytometry analyses and caspase activation assays, expression analyses

of pro-apoptotic (Bax, Bak, Puma) and

anti-apoptotic (Bcl-2, Bcl-XL) genes of Vero cell were

performed. GAPDH and Beta actin reference genes were used to normalize gene expression

levels of apoptosis related genes (Table S3). Expressions of Bak,

Bax, Bcl-2, Bcl-XL and

Puma genes of Vero cells were analyzed following SBV infection. It was

seen that increasing levels of gene expression has higher ratio in those of which are

normalized to GAPDH reference gene than those were normalized to Beta actin reference gene

(Figs. 4 and 5).

In normalization to GAPDH reference gene, Bak, Bax, and

Puma gene expressions were upregulated, whereas Bcl-2

and Bcl-XL genes were downregulated gradually by time (Fig. 4). Statistically significance

(P<0.01) is determined for 0.1 MOI infected cells in comparison to

mock infected group for Bak (at 2, 6, 12, 18, 24, and 36 hr pi),

Bax (at 24 and 48 hr pi), Puma (at 48 hr pi),

Bcl-2 (at all time points) and Bcl-XL (at all time

points) genes. In cells infected with 0.01 MOI of SBV, there is statistically significance

(P<0.01) in different MOI doses and in between time points and mock

infected group. Statistically significance (P<0.01) is determined in

GAPDH normalization for 0.01 MOI infected cells in comparison to mock infected group in

Bak (at 2, 12, 18, 24, 36 and 48 hr pi), Bax (at 6 and

48 hr pi), Puma (at 36 hr pi), Bcl-2 (at 2, 6, 24, 36,

and 48 hr pi) and Bcl-XL (at 2, 12, and 48 hr pi) genes.

In normalization to Beta actin reference gene, only Puma gene expression

was upregulated (Fig. 5). There is statistically

significance (P<0.01) determined for both 0.1 MOI and 0.01 MOI

infected cells at all time points in comparison to mock infected group for

Bak, Bcl-2 and Bcl-XL genes, and for

Bax gene at 6, 12, 18, 24, 36, and 48 hr pi. For Puma

gene, the cells infected with 0.1 MOI (at 18 and 48 hr pi) and 0.01 MOI (at 2, 6, 18, 24,

36, and 48 hr pi) have shown statistically significance (P<0.01) and

when compared to mock infected group. The Beta actin normalization results demonstrated

that SBV causes upregulation of Puma gene in Vero cells, however does not

increase the levels of pro-apoptotic genes Bak and Bax

and SBV infection culminated in downregulation of anti-apoptotic genes

Bcl-2 and Bcl-XL.

According to both GAPDH and Beta actin reference genes, Bcl-2/Bax rate was 0.19 and 0.16

in 48 hr pi in 0.1 and 0.01 MOI infections, respectively. Similarly, Bcl-XL/Bax rates were

identical when calculated according to GAPDH and Beta actin reference genes. Rate of

Bcl-XL/Bax at 48 hr pi was 0.88 and 0.45 for 0.1 and 0.01 MOI infections,

respectively.

DISCUSSION

Cell death in viral infections could be directed by some viral proteins both for induction

and inhibition of apoptosis. Inhibition of apoptosis by certain viruses can be related with

persistent infection and achievement of viral replication. Some viruses which induce

apoptosis in cells could be for lytic infection and viral spread without any inflammation

and immune response. To date, some members of Peribunyaviridae family were

found to induce apoptosis in several cell lines. Akabane, Aino, Crimean-Congo hemorrhagic

fever, Oropouche viruses of Peribunyaviridae family reported as triggering

apoptosis in different cell lines such HeLa, Huh7 and Vero [16, 24]. SBV, another member of

Peribunyaviridae, was shown to induce apoptosis in CPT-Tert and HEK-293T

cell lines but there was no data about induction

or inhibition of apoptosis by SBV in different cell lines except these. This lack of

information on Vero cell line, one of the most preferred and susceptible cell line for SBV

was eliminated by data of this study.

Induction of apoptosis could be detected by many features of cells in

vitro. DNA fragmentation, caspase activation, cleavage of apoptosis related

proteins such as caspases, PARP etc., translocation of cytochrome c from mitochondria to

cytoplasm, increasing of expression of pro-apoptotic genes, phosphatidylserine exposure on

outer leaflet of cells are the most reliable and commonly used characteristics of apoptotic

cells and could be detected in vitro by using different methods. In present

study, DNA fragmentation, phosphatidylserine exposure, caspase activation and apoptosis

related gene expression levels were analyzed with agarose gel electrophoresis, Annexin V/PI

staining in flow cytometry, colorimetric caspase activation assays and real-time PCR

methods, respectively.

DNA fragmentation is one of the basic characteristics of apoptosis/necrosis and can be

detected in agarose gel electrophoresis. Results of this study on DNA fragmentation of SBV

infected Vero cells was detected as being dependent on infection time and dose of virus.

First DNA fragmentation of SBV infected cells were seen on 18 hr pi at 0.1 MOI and on 24 hr

pi at 0.01 MOI of SBV (Fig. 1). Virus dose and

infection time can affect the first DNA fragmentation appearance on virus-infected cells. A

detectable DNA laddering at the 2 MOI of H9N2 virus infected A549 cells was detected at

16 hr pi whereas all 0.1, 1 and 10 MOI of

chikungunya virus infected cells showed DNA fragmentation at 48 hr pi.

Cells under physiological conditions have phosphatidylserine on inner leaflet of plasma

membrane. During apoptosis induction in cells phosphatidylserine become exposed on outer

leaflet of plasma membrane and display “eat me” signal to phagocytes. Annexin V, an anticoagulant protein which binds to

phosphatidylserine, is used for detection of apoptosis incidence in cells. Propidium iodide

(PI) which is used widely with combination of Annexin V is a plasma membrane permeability

marker. When used combined together cells could be seperated into 4 groups: live (Annexin

V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+) and necrotic

(Annexin V−/PI+) cells. Flow cytometry results

showed that highest apoptotic cell rate were in 48 hr pi samples in infection of 0.1 MOI

(Fig. 2A). Early apoptosis rates were 24.04% and

17.1% while late apoptosis rates were 13.8% and 5.6% in 0.1 and 0.01 MOI of SBV infected

cells, respectively (Fig. 2). This is the first

results of apoptosis detection of SBV infected Vero cells by using Annexin V/PI staining in

flow cytometry. Apoptotic cell percentage could be affected by virus species/strain, virus

dose, cell line and time of infection. Early and late apoptotic cells were detected as 61–75

and 13–19%, respectively, in HeLa cells transfected with canine parvovirus-2 NS1 encoding

vector. It is surprising that phosphatidylserine

externalization starts in the early time in the flow cytometry results but it is not seen

caspase-3 activation until 36 hr pi.

Caspases, cystein proteases that modulates apoptosis, cleave their specific substrates and

activate downstream molecules and apoptotic cell death occurs via mainly two pathways:

extrinsic and intrinsic pathways. In present study, caspase-3 which is one of the effector

caspases, caspase-8 and caspase-9 which have roles on initiation of extrinsic and intrinsic

pathways respectively had been studied on Vero cells infected with SBV. To date whether SBV

leads to caspase activation of infected cells had investigated in only CPT-Tert and HEK-293T

cells with result of slight activation of caspase-3/7. Paucity of information in Vero cell line for caspase activation has eliminated

in present study with caspase-3 activation and further investigation of pathway related

caspases. Caspase-3 was activated following SBV infection in both MOIs, but statistically

significance is determined for 36 and 48 hr pi cells compared to mock infected group

(P<0.01), confirming that SBV induces apoptosis in Vero cells (Fig. 3).

To determine whether SBV modulates extrinsic and intrinsic signaling cascade, caspase-8 and

caspase-9 activation was analyzed, respectively. Caspase-8 was slightly but significantly

(P<0.01) activated in SBV infected cells. However, unexpectedly, at 2,

6, 12 and 18 hr pi of 0.01 MOI proportions of activated caspase-8 are higher than 0.1 MOI

groups (Fig. 3). Caspase-8 activation in cells at

0.1 MOI initiated from 18 hr pi and reached maximum level at 48 hr pi. Activated caspase-8

levels were slightly high at first hours of infection and interestingly peaked at 18 hr pi

at 0.01 MOI (Fig. 3). Caspase-9 activation of SBV

infected cells was investigated to find out if intrinsic pathway is triggered. Likely

caspase-8, activation of caspase-9 at 0.1 MOI has reached to highest level at 48 hr pi. Cell

group infected with 0.1 MOI of SBV has higher proportion of activated caspase-9, while

activation of caspase-9 indicated moderate but significant increases at 0.01 MOI and

interestingly, stayed almost unchanged during the time (Fig. 3). These data indicate that SBV induce intrinsic cascade. Crimean-Congo

hemorrhagic fever virus (CCHV) which is another member of family

Peribunyaviridae induces apoptosis via both extrinsic and intrinsic

pathways.

Many research reported that pro-apoptotic genes like Bak,

Bax and Puma are upregulated when apoptosis induced in

cells. Bak gene was upregulated in Hepatitis C virus infected tissues and

HepG2 cell lines associated with apoptosis induction. Chicken fibroblast cells infected with infectious bronchitis virus (IBV)

showed increased Bak gene levels as 1.39 and 2.09 fold after 8 and 16 hr

pi, respectively. Bak levels were 6.41 fold increased in IBV infected

chicken embryos after 48 hr pi. In this study,

at 48 hr pi Bak gene levels normalized to GAPDH were found increased

approximately as 0.93 fold in 0.1 MOI and 1.40 fold in 0.01 MOI (Fig. 4), whereas levels normalized to Beta actin were found

approximately 0.25 fold in 0.1 MOI and 0.14 fold in 0.01 MOI (Fig. 5). In normalization to GAPDH reference gene,

Bak gene expression was upregulated, whereas the gene was downregulated

according to Beta actin normalization. These data indicates that SBV infection has minor

effect on Bak gene. Although certain viruses can affect or interact with

Bak [32, 35],

there is no study that investigate whether SBV interact with Bak protein in cellular level.

Bax gene expression level was found to be increased in canine distemper

virus infected dog’s cerebellum as approximately 9.8 fold. According to GAPDH normalization in this study Bax gene were

increased approximately 1.47 fold in 0.1 MOI and 3.02 fold in 0.01 MOI of SBV in 48 hr pi in

Vero cells (Fig. 4). Bax gene was

upregulated as 0.39 fold in 0.1 MOI and 0.30 fold in 0.01 MOI in 48 hr pi of SBV infection

when expression level was normalized to Beta actin (Fig.

5).

Bcl-2 gene which is an anti-apoptotic gene blocks apoptosis induction in

healthy cells. When apoptosis is triggered in cells Bcl-2 gene expression

starts to decrease. Bcl-2/Bax rate in West Nile virus infected Neuro2a cells were detected

as 3.50 in 0 hr and 0.40 in 6 hr pi. In this

study Bcl-2/Bax rate was 0.82 in 2 hr pi and 0.19 in 48 hr pi in 0.1 MOI of SBV, whereas

0.36 in 2 hr pi and decreasing to 0.16 in 48 hr pi in 0.01 MOI, according to both

normalization with GAPDH and Beta actin, indicating that SBV induce apoptosis by affecting

apoptosis related genes. Another anti-apoptotic gene Bcl-XL inhibits

mitochondrial permeabilization in cells and prevents apoptosis. Bcl-XL/Bax rate in 0.1 MOI

of SBV infected Vero cells was 0.86 in 2 hr pi and 0.88 in 48 hr pi. For 0.01 MOI,

Bcl-XL/Bax rate was 0.48 in 2 hr pi and decreasing to 0.45 in 48 hr pi. Bcl-XL/Bax rate was

same with both GAPDH and Beta actin normalizations and results indicated that SBV does not

affect Bcl-XL/Bax rate significantly.

Puma is pro-apoptotic gene which has critical role on apoptosis induced by

wide range of stimuli such as toxins, endoplasmic reticulum stress, ischemia and infections.

CCHV causes upregulation of Puma gene in Huh7 cell line at both 1 and 0.1

MOI. Puma is found to be

induced in HIV envelope transfected HeLa cells.

In present study Puma is upregulated in Vero cells at the highest level in

comparison to other pro-apoptotic genes investigated (Figs. 4, and 5). In GAPDH normalization

statistically significance (P<0.01) is determined for 0.1 MOI and 0.01

MOI infected cells in comparison to mock infected group at 48 hr pi and 36 hr pi,

respectively, for Puma (Fig. 4).

The results of Beta actin normalization indicated that the cells at 18 and 48 hr pi of 0.1

MOI and the cells at 2, 6, 18, 24, 36, and 48 hr pi of 0.01 MOI have shown statistically

significance (P<0.01) when compared to mock infected group, causing

upregulation of Puma gene in Vero cells (Fig. 5). These results are thought to be related with apoptosis of Vero cells by

SBV could be induced dominantly via Puma gene, as given that Rift Valley

fever virus (RVFV), another member of Peribunyaviridae, is shown to use p53

pathways and cause upregulation of Puma. Activation and/or utilization of p53 pathways upon SBV infection still remain

unclear.

This is the first detailed study of apoptosis induction in Vero cells caused by SBV

infection. Results of this study showed that SBV induce apoptosis via both intrinsic and

extrinsic pathways and moreover intrinsic pathway induction could be modulated over

Puma gene. To understand the whole molecular mechanisms used by SBV in

both Vero and other cell lines, more detailed studies should be performed in future.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.