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ANTIVIRAL ACTIVITIES OF… Sunmola et al., FJS
FUDMA Journal of Sciences (FJS) Vol. 3 No. 3, September, 2019, pp 540 - 545
540
ANTIVIRAL ACTIVITIES OF SUPERNATANT OF FERMENTED MAIZE (OMIDUN) AGAINST SELECTED
ENTEROVIRUSES
1Sunmola, A. A., *2Ogbole, O. O., 3Faleye, T. O. C., 2Adeniji, J. A. and *1Ayeni, F. A.
1Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ibadan, Ibadan, Oyo State, Nigeria
2Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Oyo State, Nigeria
3Department of Virology, University College Hospital, University of Ibadan, Ibadan, Oyo State, Nigeria
*Corresponding authors email: nikeoa@yahoo.com, funmiyeni@yahoo.co.uk
ABSTRACT
Enteroviruses (EVs) are of enormous public health significance being the etiological agents of an array of
clinical conditions, local fermented product may confer protection in the gastrointestinal tract against EVs.
Omidun (supernatant of fermented maize) has been traditionally used to reduce the frequency of stooling
during diarrheal episodes. However, there is no information on the antiviral activities of Omidun on EVs and
the scientific proof of the traditional claims. This study, therefore, investigates the antiviral potentials of
Omidun against EVs. The antiviral activity of Omidun was determined against Echovirus 7 (E7), E13 and
E19 in a pre- and post-treatment approach. The antiviral effect was determined by cytopathic effect and cell
viability using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor. (MTT) assay. Omidun had
high percentage inhibition for E7 and E19 with 83.5% and 77.9% respectively, in pre-treatment, and 78.2%
and 77.5% respectively, in post-treatment. Minimal effects were observed for E13; 12.2% and 24.2% in pre-
and post-treatment, respectively. Generally, antiviral activity for pre-treatment was better than post-
treatment. Omidun was shown to have the potential to inhibit the replication of selected EVs. Thus,
supporting the traditional claims of its therapeutic effect. To the best of our knowledge, this is the first report
of antiviral activities of Omidun.
Keywords: Omidun, Human Enterovirus and Antiviral
INTRODUCTION
Enterovirus (EV) is a genus in the Picornaviridae family (Tan
et al., 2011) and poliovirus (the etiological agent of
poliomyelitis) is the prototype members of the genus. The
elimination of poliovirus through vaccination in many parts
of the world and the ultimate eradication (in the near future)
of the virus globally has resulted in non-polio EVs (NPEV)
taking center stage as agents of public health significance.
This is manifest in the emergence of EV-D68 and EV-A71 as
the leading etiological agents of EV associated neurological
manifestations like Acute Flaccid Myelitis (AFM); a clinical
condition very similar to poliomyelitis and seems to be taking
its niche) globally (Bitnun et al.,2018). Considering the
public health significance of NPEVs and the dearth of
vaccines and chemotherapeutics agents, there is need for
investigation of alternative strategies for NPEV control.
Enteroviruses are spread mainly via the faecal-oral route and
the gastrointestinal tract serves as their site of primary
replication (Nathanson et al., 2010; Tapparel et al., 2013).
Lactic acid bacteria (LAB) are very effective in the treatment
of gastrointestinal diseases (Pant et al., 2007; Bamidele et al.
2013; Bamidele et al. 2014; Sunmola et al. 2019) and they
naturally reside in fermented foods. In Nigeria, one very
common indigenous fermented food with functional lactic
acid bacteria is Ogi. It is an acidic fermented cereal mash
formulated from maize. It is usually prepared as a smooth
porridge with sour taste and redolent similar to yoghurt
(Falana et al., 2011) and serve as staple meal for
convalescences and the aged as well as popular weaning food
for infants [Teniola and Odunfa 2001; Omemu et al., 2011].
Omidun is the watery supernatant of “Ogi” and has been
traditionally found to be of medicinal importance in the
South-Western part of Nigeria. It is used as solvent for herbal
extraction especially to treat malaria and fevers (Falana et al.,
2016). Omidun is also used traditionally for the control of
diarrhoea and other GIT disorders ([Teniola and Odunfa
.2001). Microorganisms, especially LAB in Omidun and other
indigenous fermented foods have been reported to have
antimicrobial efficacy against pathogenic microorganisms
(Ayeni et al., 2006; Ayeni et al., 2009; Ayeni et al., 2011;
Falana et al., 2012; Afolayan and Ayeni 2017). However, in
spite of traditional claims of the therapeutic efficacy of
Omidun against GIT disorders, there is no report of antiviral
activities of Omidun against EVs. Thus, in this study, we aim
to examine the in vitro antiviral activity of Omidun against
three NPEVs (Echovirus 7 [E7], E13, and E19) commonly
recovered from acute flaccid paralysis cases in Nigeria
METHODOLOGY
Cultures of cell line
Human rhabdomyosarcoma (RD) cells were maintained in
Dulbecco’s modified eagle media (DMEM) supplemented
with 10 percentage v/v heat-inactivated foetal bovine serum
(FBS). Cells were detached from flask (trypsinized) using
0.25% v/v trypsin. Subsequently, growth medium (DMEM
supplemented with 10% FBS) was used to terminate
trypsinization and 100 µL of medium with cells were
FUDMA Journal of Sciences (FJS)
ISSN online: 2616-1370
ISSN print: 2645 - 2944
Vol. 3 No. 3, September, 2019, pp 540 –545
ANTIVIRAL ACTIVITIES OF… Sunmola et al., FJS
FUDMA Journal of Sciences (FJS) Vol. 3 No. 3, September, 2019, pp 540 - 545
541
transferred into 96 wells plate, then, incubated in humidified
incubator at 37⁰C, 5% Carbon dioxide for 24 hrs. The cells
used were passaged between 7 to 10 times throughout the
study.
Viruses
Three echoviruses (E7, E13 and E19) previously recovered
from acute flaccid paralysis cases (Faleye et al., 2017) were
passaged in RD cell line (UCH-WHO group code RD/P231).
They were freeze-thawed three times, centrifuged and the
supernatant was aliquoted into 500-µl cryovials and stored at
− 20 C until use.
Virus quantification and infection titre calculation
A method similar to that used by (Wei et al., (2014) was used
to quantify the Echoviruses used in this study. One hundred
microliters (100 µL) of RD cell suspension was seeded into
each well of a 96 well plates as previously described. After 24
hours incubation, 100 μl of 10-fold serially diluted viral
suspension was added and then incubated till the virus control
wells had developed 100% cytopathic effects (CPE) after 24
h. The CPE were scored visually using an inverted
microscope and the virus infection titers were expressed as
Tissue Culture Infective Dose (TCID50). Viral titers were
determined using the standard method of median tissue
culture infective dose (TCID50) on the cells. Infectious titer
was calculated using Kerber Spearmans formular.
Preparation of Omidun
Fresh Ogi was prepared as previously described by Afolayan
et al. (2017). The whole mass was re-suspended in water and
allowed to ferment for 24 hours. The supernatant (Omidun)
was obtained. The maximum number of bacteria (Minimum
Cytotoxic Concentration; MCC) introduced into the antiviral
assays was determined by titration (cells exposed to different
Omidun concentrations) against the cell line; a process similar
to cell cytotoxic concentration (CC50) by Wei et al., (2014).
The MCC was carried out by preparing twelve (12) 1:2 serial
dilutions of the Omidun. Each dilution was inoculated (in
eight wells) into the monolayer of cells in 96 well plate.
Dilution 11, found to be the dilution with the least viable cells,
was plated out on de Mann Rogosa Sharpe (MRS) agar to
confirm presence of LAB which gave a count of 36.
Antiviral effect: Pre treatment
The antiviral activities of Omidun against E7, E13 and E19
was determined according to inhibition of virus-induced CPE
in acutely infected RD cells. Confluent RD cell monolayer in
96 well plate was inoculated with 50 µL of Omidun in
triplicates and allow to adsorb for 90 mins after which 50 µL
of the respective viruses (E7, E13 and E19) containing 100
TCID50 were added (Khania et al., 2012). The preparation
was sealed and incubated in humidified incubator at 37⁰C, 5%
carbon dioxide for 48 hrs. CPE was observed microscopically
and the viability of the cells was determined using MTT
assays. .
Antiviral effect: Post treatment
The antiviral activities Omidun against E7, E13 and E19 was
determined according to inhibition of virus-induced CPE in
acutely infected RD cells. Confluent RD cell monolayer in 96
well plates was infected with 50 µL of virus (E7, E13 and
E19) containing 100 TCID50 and allowed to adsorb for 40
mins for pretreatment and 40 minutes for post treatment after
which 50 µL of Omidun was added (Khania et al., 2012). The
preparation was sealed and incubated in humidified incubator
at 37⁰C, 5% carbon dioxide for 48 hours. The CPE was
observed microscopically and the viability of the cells was
determined using MTT assays.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromidefor (MTT) assay
The MTT assay (Sigma – USA) was used to quantify living
cells via mitochondrial metabolism. The assay was carried out
according to slightly modified method of Khania et al.,(2012).
After the incubation period (48 h at 37°C) in presence of 5%
carbon dioxide on a 96-well plate, the medium was removed
and 25 μl of ready MTT dye was added. The plate was
incubated at 37°C for 4 hrs. After this period, 100 μl of
dimethyl sulphoxide (DMSO) was added; the contents were
shaken for few minutes and the optical density of each well
was determined with a microplate reader (Bio-Tek USA) at
490 nm.
Data analysis
The percentage inhibition was calculated with the mean
optical density using Microsoft Excel software with the
formula below;
Virus control – Test × 100%
Virus control
Optical densities of the MTT assay of the treatment and
control wells in vitro anti-viral study were obtained and all
results were expressed as means ± SD (Standard deviation of
mean). Differences between means across groups were tested
for statistical significance using a one-way analysis of
variance (ANOVA) with the Tukey post hoc test. All
statistical analyses were carried out with Graph Pad Prism
version 5.0.
RESULTS
Antiviral activities of Omidun against E7, E13 and E19
Omidun was administered to the cells before (pre-treatment)
infection with E7, E13 and E19 (Figure 1a), The final
concentration of Omidun used that had best cell survival was
at 1.8 x 102 CFU/ml of viable bacterial cell. Omidun had a
high percentage inhibition of 83.5% and 77.9% for E7 and
E19, respectively. For E13, on the other hand, the percentage
inhibition was low with a value of 12.2%.
When Omidun was administered to the cells after (post-
treatment) infection with E7, E13 and E19 (Figure 1b),
Omidun had a percentage inhibition of 78.2%, 77.5% and
24.2% for E7, E19 and E13 respectively. As with pre-
treatment, the inhibitory effects observed were most
pronounced in the highest concentration of Omidun used.
ANTIVIRAL ACTIVITIES OF… Sunmola et al., FJS
FUDMA Journal of Sciences (FJS) Vol. 3 No. 3, September, 2019, pp 540 - 545
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Figures 1a and Ib: Percentage inhibition of Omidun for pre-treatment (a) and post-treatment (b) against EV7, EV13 and
EV19 at different dilutions.
Comparison of Pre- and Post-treatment of E7, E13 and E19 infected cells with Omidun
For E7 and E19, the proportion of viable cells was higher in pre-treatment than post-treatment (Figure 2). The difference was
however, only statistically significant (P<0.01) for E7. For E13, the proportion of viable cells was higher in post-treatment
than pre-treatment. However, there was no statistically significant difference between pre- and post-treatment with Omidun
against E13 and E19 (Figure 2).
ANTIVIRAL ACTIVITIES OF… Sunmola et al., FJS
FUDMA Journal of Sciences (FJS) Vol. 3 No. 3, September, 2019, pp 540 - 545
543
Fig. 2: Pre-treatment and post-treatment intervention of Omidun against EV7, EV13 and EV19 infected RD cells. Data
presented as Mean ± SD <P0.01.
DISCUSSION
In this study, we have demonstrated the antiviral activity of
Omidun against selected EVs in vitro. Omidun being a vehicle
for LAB in fermentation state tend to potentiate its activity.
Lactic acid bacteria have multiple antimicrobial mechanisms
which prevent viral infection of the cells and some LAB
strains have antiviral activities which deduce a strain
dependent antiviral effect (Khania et al., 2012).
Pre-incubation approach has also been previously reported to
be better than co-incubation in prevention of pathogen
adherence due to the fact that some organisms adhere rapidly
to cells than others and any detached LAB strains are readily
replaced by surrounding pathogenic organisms (Sayyed et al.,
2013). In this study, pre-treatment was seen to be more
effective than post-treatment as displayed by Omidun against
E7. There was no significant difference between the pre-
treatment and post-treatment effect of Omidun used in this
study for E13 and E19. The replication cycle of EVs is usually
complete in six (6) hours (Carter et al., 2007). Hence, the 40
minutes delay in the post-infection administration of Omidun
(post-infection assay) should have given the viruses a head
start in the infection process and possibly attachment and
entry should have been initiated (if not completed) by the time
Omidun was administered. Hence, the observed antiviral
activity of Omidun when administered post-infection might
imply that a mechanism also exists by which some component
of Omidun can halt or interrupt (therapeutically) an already
initiated EV (at least E7 and E19) replication cycle.
Considering, E7, E13 and E19 are all members of EV Species
B (EV-B) and were recovered from AFP cases around the
same time (Faleye et al., 2017), the well documented
recombination within the non-structural genomic regions (P2
and P3) of members of the same EV Species (Nikolaidis et
al., 2019) suggests that region of their genomes would be very
similar. Hence, might not be completely responsible for the
observed resistance of E13 to Omidun while E7 and E13 were
susceptible. The switching (via recombination) of the non-
structural genomic regions (P2 and P3), restores
pathogenicity and transmissibility to the Sabin (Vaccine)
strains of the Polioviruses and thereby contribute to the
emergence of Vaccine Derived Polioviruses (Burns et al.,
2014), It may however be difficult to completely rule out the
possibility that differences in the non-structural genomic
regions (P2 and P3) of the viruses could contribute to the
different phenotypes observed in response to therapeutic
treatment of infected cells with Omidun.
The control wells containing only RD cells and Omidun had
higher values for the viability (MTT) assay compared to the
control wells that had only RD cells. This suggests that some
components of Omidun might be helping the cells grow better
or remain viable longer. The other possibility is that some of
the LAB in the Omidun could have been present in the wells
during the MTT assay and (being viable cells too) increased
the read-out for viability (Hundie et al., 2016). Considering
that these LAB might have been present in the experiment
wells containing RD cells, EVs and Omidun, it is likely that
the values for the viability (MTT) assay might have been
exaggerated by measuring both viable RD and LAB.
CONCLUSION
Omidun, was able to reduce the viral infectivity of the
Enterovirus-7 and Enterovirus -19 to a large extent and its
pre-treatment is more effective than its post-treatment.
Constant consumption of Omidun might be beneficial for its
antiviral effects.
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FUDMA Journal of Sciences (FJS) Vol. 3 No. 3, September, 2019, pp 540 - 545
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LIMITATION OF THE STUDY
The viable RD cells were not delineate from viable lactic acid
bacteria that might be present in Omidun.
COMPETING INTEREST
The authors declare that they have no competing interests.
AUTHORS’ CONTRIBUTIONS
AAS carried out laboratory studies and drafted the
manuscript. OOO designed the study and revised the
manuscript. TOCF revised the manuscript. JAA provided the
viral materials for the study, FAA designed the study and
revised the manuscript.
ACKNOWLEDGEMENTS
Our sincere gratitude goes to Mr Salawu for his support. The
technical support provided by the WHO Polio Lab, Ibadan,
Nigeria, is deeply appreciated.
REFERENCES
Adeniyi, B.A., Ayeni, F.A., Ogunbanwo, S.T. (2006).
Antagonistic activities of Lactic Acid Bacteria isolated from
Nigerian fermented dairy foods against organisms implicated
in Urinary Tract Infection. Biotechnology, 5(2), 183-188.
Afolayan, A.O., Ayeni, F.A. (2017). Antagonistic effects of
three lactic acid bacterial strains isolated from Nigerian
indigenous fermented Ogi on E. coli EKT004 in co-culture.
Acta Alimentaria, An International Journal of Food Science.
46 (1), 1–8.
Afolayan, A.O., Ayeni, F.A., Ruppitsch, W. (2017).
Antagonistic and quantitative assessment of indigenous lactic
acid bacteria in different varieties of Ogi against
gastrointestinal pathogens. Pan African Medical Journal. 27:
22.
Ayeni, F.A., Adeniyi, B.A., Ogunbanwo, S.T., Tabasco, R.,
Paarup, T., Peláez, C., Requena, T. (2009). Inhibition of
uropathogens by lactic acid bacteria isolated from dairy foods
and cow’s intestine in western Nigeria. Archives of
Microbiology 191 (8), 639-648.
Ayeni, F.A., Sánchez, B., Adeniyi, B.A., de los Reyes-
Gavilán, C.G., Margolles, A., Ruas-Madiedo, P. (2011).
Evaluation of the functional potential of Weissella and
Lactobacillus isolates obtained from Nigerian traditional
fermented foods and cow´s intestine. International Journal of
Food Microbiology. 147 (2), 97-104.
Bamidele, T.A., Adeniyi, B.A., Ayeni, F.A., Fowora, M.A.,
Smith, S.I. (2013): The antagonistic activities of Lactic acid
bacteria isolated from Nigerian salad vegetables against
methicillin resistant Staphylococcus aureus. Global Research
Journal of Microbiology. 3(1): 18 –23.
Bamidele, T.A, Adeniyi, B.A, Ayeni, F.A, Akinsinde, KA.
(2014). Identification, Antagonistic activities against Vibrio
cholerae 01 of lactic acid bacteria isolated from Nigerian
grown salad vegetables. International Journal of
Pharmaceutical Research and Bio-science; 3(3), 56-65.
Bikila, W. (2015). Lactic Acid Bacteria: Benefits, Selection
Criteria and Probiotic Potential in Fermented Food. Journal
of Probiotics Health. 3:,29.
Bitnun, A., Yeh, E.A. (2018). Acute Flaccid Paralysis and
Enteroviral Infections. Current Infectious Disease Reports.
20(9):34
Burns, C.C., Diop O.M., Sutter R.W., Kew O.M. (2014).
Vaccine-derived polioviruses. Journal of Infectious Diseases.
Suppl 1:S283-93.
Carter, J., Saunders, V.A. (2007). Virology: Principles and
Applications. John Wiley & Sons. p. 166. ISBN 978-0-470-
02386-0.
Falana, M.B., Bankole, M.O., Omemu, A.M., Oyewole,
O.B., (2011). Microorganisms associated with supernatant
solution of fermented maize mash (Omidun) from two
varieties of maize grains. Researcher. 3(7).
Falana, M.B., Bankole, M.O., Omemu, A.M., Oyewole, O.B.
(2012). Antimicrobial potentials of some selected
microorganisms associated with supernatant solution of
fermented maize mash Omidun. African Journal of
Microbiology Research. 6(19), 4095-4101.
Falana, M.B., Bankole, M.O., Omemu, A.M. (2016).
Comparative antimicrobial potentials of Omidun obtained
from yellow and white maize varieties on some diarrhoea
causing microorganisms. African Journal of Microbiology
Research. 10(27): 1018-1023.
Faleye, T.O.C., Adewumi, M.O., Japhet, M.O., David, O.M.,
Oluyege, A.O., Adeniji, J.A., Famurewa, O. (2017). Non-
polio enteroviruses in faeces of children diagnosed with acute
flaccid paralysis in Nigeria. BMC Virology Journal. 14:175.
Hundie, G.B., Woldemeskel, D., Gessesse, A. (2016).
Evaluation of Direct Colorimetric MTT Assay for Rapid
Detection of Rifampicin and Isoniazid Resistance in
Mycobacterium tuberculosis. PLoS One. 11(12):e0169188.
Khani, S., Motamedifar, M., Golmoghaddam, H., Hosseini,
H.M., Hashemizadeh, Z. (2012). In vitro study of the effect
of a probiotic bacterium Lactobacillus rhamnosus against
herpes simplex virus type 1, Brazillian Journal of Infectious
Diseases. 16(2), 129-135.
Nathanson, N., Kew O.M. (2010). From Emergence to
Eradication: The Epidemiology of Poliomyelitis
ANTIVIRAL ACTIVITIES OF… Sunmola et al., FJS
FUDMA Journal of Sciences (FJS) Vol. 3 No. 3, September, 2019, pp 540 - 545
545
Deconstructed. American Journal of Epidemiology. 172(11),
1213 – 1229.
Nikolaidis, M., Mimouli, K., Kyriakopoulou, Z., Tsimpidis,
M., Tsakogiannis, D., Markoulatos, P., Amoutzias, G.D.
(2019). Large-scale genomic analysis reveals recurrent
patterns of intertypic recombination in human enteroviruses.
Virology. .526, 72-80.
Omemu, A.M., Atanda, O.O., Ayinde, I.A., Henshaw, F.O.
(2011). Perceptions of mothers on food safety related and the
microbiological contamination of complimentary foods – A
case study in 2 rural areas in Southwestern Nigeria.
Researcher. 3(6), 60-67.
Pant, N., Marcotte, H., Brüssow, H., Svensson, L.,
Hammarström, L. (2007). Effective prophylaxis against
rotavirus diarrhea using a combination of Lactobacillus
rhamnosus GG and antibodies. BMC Microbiology. 7:86.
Sayyed, M.H.G., Mahboobeh, M.N., Mahdi, G., Zohreh, K.
Therapy. (2013). What is the most Effective Method for Host
Protection Against Enteric Pathogen. International Journal of
Entric Pathogens. 1(2): 36-42.
Sunmola, A.A, Ogbole, O.O, Faleye, T.O.C, Adetoye, A,
Adeniji, J.A, Ayeni F.A. (2019). Antiviral potentials of
Lactobacillus plantarum, Lactobacillus amylovorus and
Enterococcus hirae against selected Enterovirus. Folia
Microbiologica. 64(2): 257–264
Tan, C.Y.Q., Ninove, L., Gaudart, J., Nougairede, A.,
Zandotti, C. (2011). A Retrospective Overview of Enterovirus
Infection Diagnosis and Molecular Epidemiology in the
Public Hospitals of Marseille, France (1985–2005). PLoS
ONE. 6(3): e18022.
Tapparel, C., Siegrist, F., Petty, T.J., Kaiser, L. (2013).
Picornavirus and enterovirus diversity with associated human
diseases. Infection, Genetics and Evolution. 12(3):505-521.
Teniola, O.D., Odunfa, S.A. (2001). The effects of processing
methods on the levels of lysine, methionine and the general
acceptability of Ogi processed using starter cultures.
International Journal of Food Microbiology. 63(1-2):1-9.
Wei, Y., Fang, W., Wan, Z., Wang, K., Yang, Q., Cai, X., Shi,
L., Yang, Z. (2014). Antiviral effects against E71 of
pimprinine and its derivatives isolated from Streptomyces sp.
Virology Journal. 11:195.
