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Replication of a malaysian strain avian influenza a virus H5N1 in madin-darby canine kidney and african green monkey kidney cells

Tan TS, Sharifah Syed Hassan, Yap WB

Sains Malaysiana, 2016;45:787-793.

The use of cell lines such as Madin-Darby Canine Kidney (MDCK) and African Green Monkey Kidney (Vero) cells in
influenza vaccine production is much advocated presently as a safer alternative to chicken embryonated eggs. It is
thus essential to understand the influenza virus replication patterns in these cell lines prior to utilizing them in vaccine
production. The infectivity of avian influenza A virus (A/Chicken/Malaysia/5858/2004) H5N1 in MDCK and Vero cell
lines was first assessed by comparing the cytopathic effect (CPE) caused by the virus infection. The viral loads in both
of the infected media and cells were also compared. The results showed that both of the MDCK and Vero cells began to
exhibit significant CPE (p<0.05) after 48 h post-infection (h p.i). The MDCK cell line was more susceptible to the virus
infection compared to Vero cell line throughout the incubation period. A higher viral load was also detected in the host
cells compared to their respective culturing media. Interestingly, after reaching its maximum titer at 48 h p.i, the viral
load in MDCK cells declined meanwhile the viral load in Vero cells increased gradually and peaked at 120 h p.i. Overall,
both cell lines support efficient H5N1 virus replication. While the peak viral loads measured in the two cell lines did
not differ much, a more rapid replication was observed in the infected MDCK samples. The finding showed that MDCK
cell line might serve as a more time-saving and cost-effective cell culture-based system compared to Vero cell line for
influenza vaccine production.
Fulltext Significant replication time-points of avian influenza a virus strain H5N1 in Madin-Darby Canine Kidney cells

Tan, Toong Seng, Yap, Wei Boon, Sharifah Syed Hassan

Jurnal Sains Kesihatan Malaysia, 2016;14(1):17-21.
MyJurnal

The occasional influenza pandemics and the seasonal influenza epidemics have destroyed millions of lives since
the last century. It is therefore necessary to understand the virus replication patterns as this provides essential
information on the virus infectivity, pathogenicity and spread patterns. This study aimed to investigate the replication
of avian influenza A virus H5N1 (A/Chicken/Malaysia/5858/2004) in MDCK cells. In this study, the TCID50 (50% tissue
culture infectious dose) of AIV H5N1 was first determined. The MDCK cells were then infected with AIV H5N1 at TCID50
for 0-48 h. The CPE (cytopathic effect) was observed and cell death was determined hourly. The virus-infected cells
and media were subsequently collected for gene analysis. The results showed that the TCID50 of AIV H5N1 was 10-9
dilution. The CPE percentage showed a strong and positive correlation with the infection period (r = 1.0, n = 9, p <
0.01). The amount of a highly conserved influenza viral gene, M2 gene amplified from infected media (r = 0.471, n =
9, p= > 0.05) and infected cell (r = 0.73, n = 9, p < 0.05) were also positively correlated with the infection period. In
conclusion, although CPE started to be observed in the early time points of infection, however, the M2 gene was only
amplified from the infected media and cells after 48 h and 24 h, respectively. This signifies that AIV H5N1 used in this
study is pathogenic and it is able to cause severe cytopathology to host cells even at low virus load.
Fulltext Universal oral vaccine for influenza infections

Yap, Wei Boon, Toong, Seng Tan, Sharifah Syed Hassan, Jeffrey Cheah

Jurnal Sains Kesihatan Malaysia, 2018;16(2):51-64.
MyJurnal

Each year, influenza A infections have caused tremendous death rate as high as 300,000-500,000 globally. Although
there are effective anti-influenza agents and vaccines, high mutational rate among influenza A viruses renders dramatic
decline in the effectiveness of anti-influenza agents or vaccines in certain individuals. The situation is further complicated
by limitations in influenza vaccine production, for instance, long production period, limited vaccine capacity and lack
of cross-protection against various influenza A virus strains. To solve these issues, development of universal influenza
vaccine based on conserved antigens such as non-stuctural protein 1 (NS1) has been endeavoured. NS1 protein is highly
conserved in all influenza A virus strains known by far, produced abundantly on infected cell surfaces and responsible for
maintaining virulence. Furthermore, cytotoxic T-lymphocytes that are active against NS1 were also reported to be able
to avoid shedding of influenza in hosts. To better inhibit influenza infections, oral immunization has long been proposed
due to feasibility of this method to be implemented and safer for recipients while able to target influenza A viruses from
the entry point. Lactobacillus has been vastly studied for its roles as bacterial carrier in oral vaccine development due
to its significant probiotic properties. For examples, stimulation of immune responses in oral and airway mucosal layers,
high colonization in oral and airway mucosal layers and great natural adjuvant effects. In this light, influenza universal
oral vaccine developed using NS1 dan Lactobacillus should be further studied in influenza oral vaccine design.
Fulltext Synergistic antibacterial effect of co-administering adipose-derived mesenchymal stromal cells and Ophiophagus hannah L-amino acid oxidase in a mouse model of methicillin-resistant Staphylococcus aureus-infected wounds

Mot YY, Othman I, Sharifah SH

Stem Cell Res Ther, 2017 01 23;8(1):5.
PMID: 28114965 DOI: 10.1186/s13287-016-0457-2

BACKGROUND: Mesenchymal stromal cells (MSCs) and Ophiophagus hannah L-amino acid oxidase (Oh-LAAO) have been reported to exhibit antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). Published data have indicated that synergistic antibacterial effects could be achieved by co-administration of two or more antimicrobial agents. However, this hypothesis has not been proven in a cell- and protein-based combination. In this study, we investigate if co-administration of adipose-derived MSCs and Oh-LAAO into a mouse model of MRSA-infected wounds would be able to result in a synergistic antibacterial effect.
METHODS: MSCs and Oh-LAAO were isolated and characterized by standard methodologies. The effects of the experimental therapies were evaluated in C57/BL6 mice. The animal study groups consisted of full-thickness uninfected and MRSA-infected wound models which received Oh-LAAO, MSCs, or both. Oh-LAAO was administered directly on the wound while MSCs were delivered via intradermal injections. The animals were housed individually with wound measurements taken on days 0, 3, and 7. Histological analyses and bacterial enumeration were performed on wound biopsies to determine the efficacy of each treatment.
RESULTS: Immunophenotyping and differentiation assays conducted on isolated MSCs indicated expression of standard cell surface markers and plasticity which corresponds to published data. Characterization of Oh-LAAO by proteomics, enzymatic, and antibacterial assays confirmed the identity, purity, and functionality of the enzyme prior to use in our subsequent studies. Individual treatments with MSCs and Oh-LAAO in the infected model resulted in reduction of MRSA load by one order of magnitude to the approximate range of 6 log10 colony-forming units (CFU) compared to untreated controls (7.3 log10 CFU). Similar wound healing and improvements in histological parameters were observed between the two groups. Co-administration of MSCs and Oh-LAAO reduced bacterial burden by approximately two orders of magnitude to 5.1 log10 CFU. Wound closure measurements and histology analysis of biopsies obtained from the combinational therapy group indicated significant enhancement in the wound healing process compared to all other groups.
CONCLUSIONS: We demonstrated that co-administration of MSCs and Oh-LAAO into a mouse model of MRSA-infected wounds exhibited a synergistic antibacterial effect which significantly reduced the bacterial count and accelerated the wound healing process.
Fulltext Anti-dengue virus serotype 2 activity of tannins from porcupine dates

Pong LY, Yew PN, Lee WL, Lim YY, Sharifah SH

Chin Med, 2020;15:49.
PMID: 32467721 DOI: 10.1186/s13020-020-00329-7

Background: Dengue fever is currently endemic in tropical and subtropical countries worldwide and effective drug against DENV infection is still unavailable. Porcupine dates, which are traditionally used to treat dengue fever, might contain potential anti-dengue compounds. Two porcupine dates, black date (BD) and powdery date (PD) from Himalayan porcupine (Hystrix brachyura), were investigated for their antiviral activities against DENV-2 in vitro.
Methods: The methanol crude extracts (MBD and MPD) were prepared from the raw material of porcupine dates. The tannin-rich fractions (BDTF and PDTF) were isolated from their methanol crude extracts using column chromatography. The presence of tannins in BDTF and PDTF extracts was determined by fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) analyses. The cytotoxicity and anti-DENV-2 activities including virus yield inhibition, virucidal, virus attachment and pre-treatment assays of the extracts were examined in Vero cells.
Results: Our findings revealed that all the extracts of porcupine dates exhibited antiviral activity against DENV-2 in Vero cells. The IC50 of BDTF and PDTF were 25 µg/mL and 11 µg/mL respectively, while their methanol crude extracts demonstrated lower antiviral efficacy (IC50 ≈ 101-107 µg/mL). BDTF and PDTF also exerted a similar higher virucidal effect (IC50 of 11 µg/mL) than methanol crude extracts (IC50 ≈ 52-66 µg/mL). Furthermore, all the extracts inhibited the attachment of DENV-2 by at least 80%. Pre-treatments of cells with BDTF and PDTF markedly prevented DENV-2 infection when compared to methanol crude extracts.
Conclusion: This study suggests that porcupine dates possess antiviral properties against DENV-2, which is attributed to its tannin compounds.