Table Of ContentAntiviral evaluation of secondary metabolites derived
from actinomycetes
conjugated to silver nanoparticles
Submitted by:
Heba Ali Ali Abd El-Gaied
MSC in Microbiology and immunology
Faculty of Pharmacy, Cairo University
For the fulfillment of the Doctor Pharmacy Degree in
Pharmaceutical Science (Microbiology and Immunology)
Under supervision of:
Prof. Dr. Abd El Gawad Hashem Dr. Marwa El Rakaiby
Professor of Microbiology Lecturer of Microbiology
and Immunology and Immunology
Faculty of Pharmacy Faculty of Pharmacy
Cairo University Cairo University
Dr. Aly Fahmy Mohamed
Head of R&D Sector, the Holding company for
production of vaccines, Sera and
drugs (Vacsera)
Faculty of Pharmacy
Cairo University
(2018)
1
Abstract
Key words: Actinomycetes – Secondary metabolites – Antiviral-
Silver nanoparticles.
Virus infections pose significant global health challenges, due to the
emergence of resistant viruses. Therefore, it is imperative that safe and
conventional antiviral drugs be developed.
In the present study, Actinomycetes isolated form Egyptian soil samples
were used to investigate the possibility of acquiring antiviral activity from their
metabolites by testing against three selected virus models. Results revealed that
the sixth fraction purified from isolate (HA1) metabolites induced significant
reduction of virus infectivity titer recording 62%, 46.5% and 44.1% log reduction
in virus infectivity for HAV, HSV-2 and VSV respectively and the isolate was
identified as Streptomyces avermitilis as blasted on the NCBI database. Further
evaluation of the antiviral activity of the metabolite against the three virus models
was attempted by conjugation to silver nanoparticles.
Introduction and Aim of work
Actinomycetes are unicellular, gram-positive bacteria, which belong to the
order Actinomycetales and possessing high (G+C) ratio in their DNA (Nonpanya
and Niamsanit 2016). Actinomycetes are the strongest antagonists among
microbes. The antibiotic substances elaborated by them possess antibacterial,
antifungal, anticancer, antiprotozoal and antiviral properties. Of the ten thousand
known antibiotics produced by microbes, approximately 70% are of actinomycete
origin, in which Streptomyces account for two thirds of this percent (De Lima
Procópio et al., 2012). They are found in many habitats as streams, rivers, lake
mud, river sediments, beach sands, sponge and marine sediments; however soil is
considered their most important habitat (Gebreyohannes et al., 2013).
2
The methods used in the identification and classification of actinomycetes
are Bergey’s Manual of Determinative Bacteriology 8th edition and International
Streptomyces project (ISP) )Shirling and Gottlieb, 1966).
Nanotechnology has been used to develop nanoparticle-based targeted
drug carriers (Falanga et al., 2011). The use of nanoparticles can be extended to
develop antivirals that act by interfering with the viral infection, particularly
during attachment and entry. Silver nanoparticles have not been only studied for
their antimicrobial activity against bacteria, but have proven to be of great
potency against several types of viruses. Many researchers have proven their
activity against human imunodeficiency virus, respiratory syncytial virus, herpes
simplex virus, and monkey pox virus. Previous researches suggested that, silver
ion may attack a broad range of targets in the virus thereby lowering the
possibility to develop resistance as compared to conventional antivirals (Galdiero
et al., 2011).
Aim of work:
In the present study, Three virus types were selected to cover both DNA
and RNA viruses; Hepatitis A, Herpes simplex virus-2 and Vesicular Stomatitis
representing positive-sense single-stranded RNA virus, double-stranded, DNA
and negative -sense single-stranded RNA respectively.
The antiviral properties of silver nanoparticles were studied on the three
virus models alone and in combination with the most potent antiviral metabolites
extracted from Egyptian actinomycetes isolates, under optimized cultural
conditions.
3
Review of literature
I. Actinomycetes
Actinomycetes are considered valuable microorganisms as they have the
potency to produce economically important therapeutic compounds and
antibiotics (Arasu et al., 2016).
Of thousands known antibiotics a decade ago, seventy percent of them are
of actinomycete origin, whereas streptomyces account for two thirds of this
percentage (Janaki et al., 2016). They are found in many habitats, such as fresh
water, cold-and warm- blooded animals, composts and sea water but soil is
considered their most important habitat (Bhatti et al., 2017). Actinomycetes
belongs to the order Actinomycetales, they are unicellular, gram-positive bacteria,
possessing high (G+C) ratio in their DNA (more than 55%). Most members are
aerobic, but few of them (as Actinomyces israelii) can grow under anaerobic
conditions (Nonpanya and Niamsanit, 2016).
II. Importance of actinomycete metabolites:
Most of commercial antibiotics like neomycin, streptomycin, erythromycin
and tetracycline are extracted from actinomycetes valuable metabolites. Neomycin
reduce the risk of bacterial contamination during surgical operations, erythromycin
is also valuable in treating upper respiratory bacterial infection such as bronchitis,
pneumonia, pertussis, urinary tract and skin infections. All these useful antibiotics
are derived from actinomycetes and are considered the basis of entire
pharmaceutical industry and haves saved human lives (Ganesan, 2008).
The discovery of novel antimicrobial or antiviral agents that are extracted
from natural sources could show novel skeletons and structures for development of
pharmaceutical industry (Newman and Cragg, 2012).
Actinomycetes produce a wide range of secondary metabolites that have
various medical importances including doxorubicin and bleomycin (antitumorals);
amphotericin B and nystatin (antifungals); rapamycin (immunosuppressives),
4
avermectin B (insecticides); phosphinotricin (herbicides) and antiviral drugs
(Grasso et al., 2016).
III. Classification and identification of actinomycetes:
The method used in the identification and classification of actinomycete is
Bergey’s Manual of Determinative Bacteriology 9th edition. In addition to
(Molecular, Chemo taxonomical and Classical Approach) that are used for the
identification of actinomycetes.
a) Molecular Approach
It studies the nucleic acids and is considered the most powerful approach
through analyzing the 16S rDNA. The analysis starts by isolating DNA and
amplifying the gene coding for 16S rRNA using the polymerase chain reaction.
The purified DNA fragments is then directly sequenced using DNA sequencer in
order to determine the order in which the bases are arranged within the length of
sample (Singh, 2017; Bull et al., 2005).
b) Chemotaxonomical Approach
Chemotaxonomical approach is taxonomically valuable because it
differentiate between suborders by analyzing the cell wall composition.
Actinobacteria peptidoglycan may contain either LL- or DL-(meso)-DAP,
depending on the genus and the sugar composition. The identification of sugar
pattern plays a key role in the identification of sporulating actinomycetes which
have meso‐ DAP in their cell walls (Sharma, 2014).
c) Classical Approach
Classical approaches for classification make use of physiological, biochemical
and morphological characters. The classical method described in the identification
key by Nonomura (1974) and Bergey’s Manual of Determinative Bacteriology
(Buchanan & Gibbons, 1974) is very useful in identifying streptomycetes. These
characteristics have been commonly employed in streptomycetes taxonomy for
many years.
5
IV. Antiviral antibiotics and enzyme inhibitors produced by actinomycetes:
Actinomycetes are the most economically and biotechnologically valuable prokaryotes having the ability to produce antiviral
metabolites.
Studies of actinomycetes previous studies on extracted secondary antiviral metabolites
Title year
Fleck et al. 1980 Isolated granatomycin which is a naphthoquinone antibiotic from Strptomyces lateritus and was found to
exhibit good antimicrobial and antiviral activities.
Ohkuma et al. 1988 Isolated an antibiotic complex from the culture filtrate of Streptoalloeeichus hindustanus sp nov
(BMY- 28190). This antibiotic complex consisted of tallysomycins A and B and exhibited strong activity
towards herpes simplex virus type I (HSV-I) and weak activity against gram positive and gram negative
bacteria.
Naruse et al. 1989 Isolated a new peptide antibiotic called lanthiopeptin, which was extracted from culture broth of
streproverticillium and was found to exhibit good antiviral activity.
Tomita et al. 1991 Found five valuable actinomycetes families that exert antiviral antibiotics. These metabolites belong to
seven compounds of fluvirucins A1, A2, B1, B2, B3, B4 and B5. All the strains had meso-2, 6-diamino-
6
pimelic acid (meso-DAP) in their cell walls. Four strains were identified as Actinomadura, while the fifth
one was Nocardioform actinomycete.
Tsunakawa etal. 1992 Isolated Amycolaptosis orientalis No.Q427-8(ATCC53884) which is a new actinomycete strain from a soil
sample collected from Maharashtra state, India. It produced a complex of new antiviral antibiotics, the
quatromicins that exhibited antiviral activity against herpes simplex virus type I influenza.
Tsunakawa etal. 1992 Isolated cycloviracins (B1 and B2) which is a new antiviral antibiotic that was extracted from
Kibdelosporangium albatum No.761-7(ATCC-55061). It showed potent antiviral activity against herpes
simplex virus type I and weak activity against Gram positive bacteria.
Naruse etal. 1993 Isolated a new actinomycete strain Microtetraspora purvosatasub sp. kistane sub sp.nov. (ATCC55076)
from a soil sample collected near Krishna river in Andhra Pradesh state, India. The isolate was found to
produce new antiviral antibiotics (kistamicins A and B). It was found to possess potent activity against
influenza virus type A.
Russel et al. 1996 Isolated quinoxapetin A and B, which are novel chromodesipeptides from a Nocardiofrom actinomycete
and they are potent inhibitors of HIV-1 and HIV-2 reverse transcriptase.
7
Tanaka et al. 1997 Isolated chloropeptins I and II which are gp 120-cd4 binding inhibitors from the mycelium of a soil
actinomycete, streptomyces sp. WK-3419. Chloropeptin I is a novel compound while chloropeptin II was
identified as complestain. Chloropeptin I has synergistic activity with anti HIV- drugs such as zidovudine
(AZT), didanosine (ddl) zalcitabne (ddc) and nevirapine.
Yakugaku Zasshi 2004 Isolated a strain that belongs to Streptomyces microflavus. The strain produced at least 13 fattiviracin
derivatives (FV-1 to FV-13). Fattiviracins recorded a potent activity against enveloped DNA viruses such
as the herpes family, VZV and HSV-1, and enveloped RNA viruses such as three strains of HIV-1 and
influenza A and B viruses.
(Divakar, 2013).
8
V. Virus isolation and cell cultures
a. Cell cultures: preferred lines for virus isolation vary from virus to another
through which virus monolayers are chose. Monolayer cultures are primary,
diploid, or continuous cell lines and are maintained in the laboratory.
b. Virus Quantitation: viruses are quantitated by titration using formulas by
Reed-Muench (1938), Karber (1931) and plaquing methods to determine the
end point from titration data (Ramakrishnan, 2016).
c. Virus isolation: viruses are incapable of reproducing on artificial media cell
as they lack ribosomes and systems for synthesis of energy and proteins; they
are not capable of reproducing on artificial media. No single system will
support the growth of all viruses. Cell cultures are prepared from cells which
have been dissociated from the parent tissue by action of proteolytic enzymes
and chelating agents. Dissociated cells are then placed in a culture vessel to
adhere and replicate to form a layer of cells (monolayer); other types of cells
such as mature hemopoietic cells and cells from malignancies may replicate
in suspension without attachment. Viruses induce changes, known as the
cytopathogenic effect (CPE) of a virus which includes any alteration in
cellular characteristics when the virus is induced. Primary cells grow from
tissue mainly epithelial cells having normal diploid chromosomes of the
same number as the parent tissue. Primary cell preparations are susceptible to
a variety of human viral pathogens. Primary cell types include primary
rhesus monkey kidney (pRMK) and primary human embryonic kidney
(pHEK). Diploid cell lines maintain their diploid chromosome number
throughout serial passages; but usually die out after the 50th passage. Diploid
cell lines include human lung fibroblasts (MRC-5 or WI-38) and human
foreskin (MRHF). Continuous (heteroploid) including human cervical
carcinoma (HeLa) and human laryngeal carcinoma (HEp-2) maintain their
9
heteroploid or aneuploid chromosome numbers during repeated subculturing.
Continuous cell lines originate from malignancies and could be cultured for
indefinite passage number.
d. Vero cell lineage: Vero cells were established from the kidney tissue of an
African green monkey (AGM). They are susceptible to various types of
viruses including SV-40, simian polyoma virus, rubella virus, arboviruses
measles and adenoviruses. They are considered the suitable choice for
various types of life-threating pathogens such as Ebola haemorrhagic fever
virus, H N influenza virus, and Middle East respiratory syndrome (MERS)
5 1
coronavirus (Osada et al., 2014).
VI. RNA and DNA viruses
A virus is a small infectious agent which is capable of replicating inside
the living cells of other organisms only. Viruses can infect animals, plants and
microorganisms including bacteria (Koonin et al., 2006).
Virions are virus particles and they consist of two or three parts: (i) the genetic
material which is either DNA or RNA, carrying the genetic information; (ii) a
capsid that is a protein coat surrounding and protecting the genetic material (iii)
an envelope of lipids that surrounds the protein coat having various shapes
ranging from simple icosahedral or simple to other more complex structures
(Canchaya et al., 2003).
Viruses are classified into seven classes according to Baltimore
Scheme as viruses lack ribosomes and rRNA, so cannot be classified within the
Three Domain Classification scheme with cellular organisms.
10
Description:conventional antiviral drugs be developed. In the present study, are Bergey's Manual of Determinative Bacteriology 8th edition and International The antiviral properties of silver nanoparticles were studied on the three pharmaceutical industry and haves saved human lives (Ganesan, 2008).
