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Virus Research 190 (2014) 1–7

Contents lists available at ScienceDirect

Virus Research

j ourna l h o mepa ge: www.elsev ier .com/ locate /v i rusres

ilver nanoparticles impair Peste des petits ruminants viruseplication

itin Khandelwala, Gurpreet Kaura, Kundan Kumar Chaubeyb, Pushpendra Singha,halini Sharmac, Archana Tiwaria, Shoor Vir Singhb, Naveen Kumarb,∗

School of Biotechnology, Rajiv Gandhi Technical University, Airport Road, Bhopal, Madhya Pradesh 462036, IndiaVirology Laboratory, Division of Animal Health, Central Institute for Research on Goats, Indian Council of Agricultural Research, Makhdoom, Mathura,ttar Pradesh 281122, IndiaDepartment of Veterinary Physiology and Biochemistry, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana 125004, India

r t i c l e i n f o

rticle history:eceived 26 March 2014eceived in revised form 22 May 2014ccepted 22 June 2014vailable online 28 June 2014

eywords:

a b s t r a c t

In the present study, we evaluated the antiviral efficacy of the silver nanoparticles (SNPs) against Pestedes petits ruminants virus (PPRV), a prototype Morbillivirus. The leaf extract of the Argemone maxicanawas used as a reducing agent for biological synthesis of the SNPs from silver nitrate. The SNPs werecharacterized using UV–vis absorption spectroscopy, X-ray diffraction (XRD) and transmission electronmicroscopy (TEM). The TEM analysis revealed particle size of 5–30 nm and the XRD analysis revealed theircharacteristic silver structure. The treatment of Vero cells with the SNPs at a noncytotoxic concentration

ilver nanoparticlesNPseste des petits ruminantsPR virusntiviral activity

significantly inhibited PPRV replication in vitro. The time-course and virus step-specific assays showedthat the SNPs impair PPRV replication at the level of virus entry. The TEM analysis showed that the SNPsinteract with the virion surface as well with the virion core. However, this interaction has no direct viru-cidal effect, instead exerts a blocking effect on viral entry into the target cells. This is the first documentedevidence indicating that the SNPs are capable of inhibiting a Morbillivirus replication in vitro.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Peste des petits ruminants (PPR) is an acute, highly infectiousisease primarily of goats and sheep (Gibbs et al., 1979). PPR

eads to high morbidity and mortality (up to 100%) resulting ineavy economic losses (Baron et al., 2011). The etiological agent,PR virus (PPRV) belongs to the genus Morbillivirus of the fam-ly Paramyxovirideae (Gibbs et al., 1979). PPR is currently endemicn Asia and Africa (Chan, 2010). Though a live attenuated vaccine isommercially available against PPR, due to its instability in subtrop-cal climate and insufficient coverage, the disease control programas not been a great success. Further, emerging evidence of poorross neutralization between vaccine strain and the PPRV strainsurrently circulating in the field has raised concerns about the pro-ective efficacy of the existing PPR vaccines (Kumar et al., 2013).

oreover, there is no antiviral therapeutics available to provide

nstantaneous protection and to reduce the risk of virus trans-

ission to the in-contact susceptible animals during epidemics.ntiviral agents could be used either to bridge the period between

∗ Corresponding author. Tel.: +91 8171301889; fax: +91 565 2763246.E-mail address: naveenkumar.icar@gmail.com (N. Kumar).

ttp://dx.doi.org/10.1016/j.virusres.2014.06.011168-1702/© 2014 Elsevier B.V. All rights reserved.

vaccination and full immunity or as an independent control mea-sure (Goris et al., 2008; Kumar and Maherchandani, 2014) andhence may be used to avoid unnecessary preemptive culling duringacute viral infections of livestock (Charleston et al., 2011). Devel-opment of novel anti-PPRV therapeutics is therefore urgent.

Modifications for potentiation of the existing antiviral com-pounds and development of novel antiviral agents are a primearea of research. Silver in different forms (metallic silver, silvernitrate, silver sulfadiazine) has been used to treat several bacterialinfections, burns and wounds since ancient time (Rai et al., 2009).Nanotechnology provides a good platform to modify properties ofthe pure metals by converting them into their nano form (nanopar-ticles) for improved biological and chemical properties (Lara et al.,2010a). The nanoparticles have the potential to be used in manydifferent medical and biological applications (Jahn, 1999; Rai et al.,2009).

Recent research has shown that silver nanoparticles (SNPs)can have effective antiviral properties against number of virusessuch as monkeypox virus (Rogers et al., 2008), Tacaribe virus

(Speshock et al., 2010), human immunodeficiency virus type 1(HIV-1) (Elechiguerra et al., 2005; Lara et al., 2010a,b), hepatitisB virus (Lu et al., 2008), respiratory syncytial virus (Sun et al.,2008), herpes simplex virus 1 (HSV-1) (Baram-Pinto et al., 2009)

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nd influenza viruses (Xiang et al., 2013; Mori et al., 2013). In thistudy we have evaluated the in vitro antiviral efficacy of the SNPsgainst PPRV, a prototype Morbillivirus.

. Materials and methods

.1. Synthesis of the SNPs

The SNPs were biologically synthesized as described previouslySingh et al., 2010). Briefly, 10 ml of aqueous leaf extract of thergemone mexicana (5 g/100 ml) was mixed with 5 mM AgNO3 solu-ion and kept at room temperature (25 ◦C) for 4 hours (h) followedy centrifugation at 10,000 rpm for 10 min. The supernatant wasiscarded and the pellet was washed twice with distilled water7000 rpm for 2 min) and allowed to dry in vacuum dryer. The driedellet was stored at room temperature for various analyses.

.2. Ultraviolet–visible (UV–vis) spectrophotometry

Bioreduction of Ag+ in the reaction mixture was monitoredy measuring UV–vis spectrum of the medium on PerkinElmer’sAMBDA 25 UV–vis spectrophotometer. The samples werecanned for determining the wavelength which showed maximumbsorbance. Milli Q water was taken as a reference. The spectrumas scanned at a resolution of 200–800 nm.

.3. Transmission electron microscopy (TEM)

The sizes of the SNPs were determined by the transmission elec-ron microscopy (TEM) (JEM-1400 VERSION 1.0). Samples wererepared by drop-coating the SNPs on carbon-coated copper TEMrids. The films on the TEM grid were allowed to stand for 2 minnd the extra solution was removed using a blotting paper. The gridas dried prior to the measurements.

.4. XRD analysis

The structure and composition of the purified (dried) SNPs wasetermined by the X’Pert Pro x-ray diffractometer (XRD) (PANalati-al empyrean – 2012) operated at a voltage of 40 kV and a current of0 mA with Cu K� radiation in �–2� configurations. The crystalliteomain size was calculated from the width of the XRD peaks usingcherrer equation by assuming that they are free from non-uniformtrains.

= 0.94�

cos �

here D is the average crystallite domain size perpendicular tohe reflecting planes, � is the X-ray wavelength, is the full widtht half maximum (FWHM), and � is the diffraction angle. To elimi-ate additional instrumental broadening, the FWHM was correctedsing the FWHM from a large grained Si sample.

corrected = (FWHM2sample − FWHM2

si)1/2

This modified equation was considered valid if the crystalliteize was smaller than 100 nm.

.5. Cells and viruses

Vero (African green monkey kidney epithelial) cells were grown

n Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma–Aldrich,t. Louis, USA) supplemented with 10% fetal bovine serumSigma–Aldrich, St. Louis, USA). PPRV/Nanakpur/2012 was prop-gated in Vero cell as described previously (Kumar et al., 2013).

search 190 (2014) 1–7

2.5.1. Determination of the cytotoxicity (MTT assay)The cytotoxicity was assessed as described previously (Kumar

et al., 2008). Briefly, Vero cell monolayers were grown in96-well plates and incubated with two-fold serial dilutionsof the SNPs or vehicle-control (DMEM), in triplicates, in atotal of 100 �l growth medium for 48 h. 20 �l of freshlymade 5 mg/ml MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] solution was added to each well, andthe cells were incubated at 37 ◦C for 4 h. The medium with MTTsolution was removed, and the formazan crystals were dissolvedwith dimethylsulfoxide. The absorption values were measured at570 nm and % cell viability was determined.

2.5.2. Efficacy of the SNP against PPRVThe SNPs were diluted in DMEM for various virological assays.

The confluent monolayers of Vero cells, in triplicates, were infectedwith PPRV at multiplicity of infection (MOI) of 0.1 in presence ofvarious concentrations of the SNPs or vehicle control. The progenyvirus particles released in the supernatants at 24 h post-infection(hpi) were quantified by plaque assay (Kumar et al., 2013) andexpressed as plaque forming unit per milliliter (PFU/ml).

2.5.3. Stability of the SNPsAliquots of the SNPs (300 �g/ml) were pre-incubated at 37 ◦C up

to 7 days and their anti-PPRV efficacy were then evaluated in Verocells, similarly as described under Section 2.5.2.

2.5.4. Virus attachment assayThe attachment assay was performed as described previously

(Kumar et al., 2011a). Briefly, Vero cells were pre-incubated withthe SNPs or vehicle control for 1 h and then infected with the PPRVat MOI of 5 for 2 h at 4 ◦C. The cells were then washed 6 times withphosphate buffered saline (PBS) and the cell lysates were preparedby rapid freeze–thaw method. The virus titers were determined byplaque assay.

Additionally, a hemagglutination inhibition (HAI) assaywas carried out as described previously (Jones et al.,2006). Heat-inactivated hyper immune serum (HIS) againstPPRV/Nanakpur/2012 was used as a positive control that inhibitsattachment of the PPRV to the host cells. 100 �l of the two-foldserial dilutions of the HIS (starting from 1/8) and the SNPs (startingconcentration 300 �g/ml; highest non-cytotoxic concentration)were incubated with equal volume of PPRV (∼106 PFU/ml) for 1 hat 37 ◦C. Thereafter, 0.5% suspension of the chicken red blood cells(RBCs) was applied and the plates were incubated at 4 ◦C for 2 h.The HAI titers were expressed as the reciprocal of the highestserum dilution that resulted in complete inhibition of hemagglu-tination. A titer of 4 was assigned if no inhibition was observed atdilution of 1:8. Results were expressed as %hemagglutination ascompare to the virus-alone sample (100% agglutination).

2.5.5. Determination of the virucidal activityThe virucidal activity was determined as described previ-

ously (Kumar et al., 2011b). Briefly, virus suspensions containingapproximately ∼106 PFU of PPRV/Nanakpur/2012 were incubatedin serum-free medium containing either DMEM or five-fold serialdilutions of the compounds for 90 min at 37 ◦C. The mixed sampleswere chilled on ice and diluted to 10−3, 10−4, and 10−5 fold beforeadding to the Vero cells for plaque assay. Plaques were stainedwith crystal violet solution and the results were plotted as %relativeinfectivity against the concentrations of the SNPs used.

2.5.6. Virus entry assayThe effect of the SNPs on PPRV entry was assessed using a previ-

ously described assay (Jones et al., 2006). The Vero cell monolayerswere prechilled to 4 ◦C and infected with PPRV at MOI of 5 in SNPs

N. Khandelwal et al. / Virus Research 190 (2014) 1–7 3

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ig. 1. Characterization of the SNPs (a). UV–vis spectra analysis of the SNPs at 4 h

NPs.

ree medium for 1 h at 4 ◦C to permit attachment. The cells werehen washed 5 times with chilled PBS to remove unattached virusnd overlaid with medium containing the SNPs or vehicle control.ntry was allowed to proceed at 37 ◦C for 1 h after which the cellsere washed again with PBS to remove any extracellular virus and

ncubated with cell culture medium without any inhibitors. Theell culture supernatants were harvested at 9 hpi for titration ofhe progeny virus particles in the treated and untreated cells.

.5.7. SNPs and PPRV interactionThe TEM analysis was performed to demonstrate the interac-

ion between SNPs and PPRV. 400 �l of PPRV (∼106 PFU/ml) werereated with 200 �l of SNPs (300 �g/ml) at room temperature. After0 min, a 10 �l droplet was deposited on a carbon-coated nickelEM grid and exposed to a 2.5% solution of PBS/glutaraldehydeapors for 30 min. The TEM observations were performed on JEM-400 VERSION 1.0.

. Results

.1. Synthesis and characterization of the SNPs

Initial aqueous solution of silver nitrate exhibited transparentolor which turned yellowish-brown following addition of the plantxtract and hence indicated the start of the formation of the SNPs.he color changed from yellowish-brown to brown within 5 min.

fter 4 h, a complete dark brown color was obtained which indi-ated the total reduction of silver ion and hence formation of theNPs. Reduction of Ag+ in the reaction medium gives an absorbanceeak between 420 and 450 nm (Li et al., 2007; Pareek et al., 2012;

ddition of plant extracts (b). TEM analysis of SNPs (c). XRD pattern of the purified

Singh et al., 2010). In UV–vis spectroscopy, we also observed anabsorbance peak at 444 nm further confirming the formation of theSNPs in the reaction medium (Fig. 1a).

The TEM analysis revealed the particle (SNPs) size, ranged from5 to 30 nm (Fig. 1b). The typical XRD pattern revealed a mixed-phase (cubic and hexagonal) structures of the SNPs with the averageestimated particle size of 20 nm (10–50 nm range), derived fromthe FWHM of peak corresponding to 1 1 1 planes (Fig. 1c). The XRDpattern showed three intense peaks in the whole spectrum of 2�value ranging from 10◦ to 80◦. A number of Bragg reflections with2� values of 32.514◦, 38.529◦, and 64.526◦ sets of lattice planes wereobserved which may be indexed to (1 1 1), (1 1 2), and (1 1 0) facetsof silver respectively.

3.2. In vitro efficacy of the SNPs against PPRV

In order to determine the in vitro efficacy of the SNPs againstPPRV, we first determined its cytotoxicity in cultured Vero cellsby MTT assay. As shown in Fig. 2a, SNPs at concentrations of≤333.33 �g/ml did not affect the cell viability even when incubatedwith the Vero cells for 72 h. At higher concentrations (1000 �g/ml),however, it was quite toxic to the cells. A non-cytotoxic concen-tration (300 �g/ml) of the SNPs was used thereafter for variousassays.

To determine the in vitro efficacy of the SNPs against PPRV, wemeasured the yield of infectious PPRV in the presence of SNPs con-

centration ranging from 1.23 to 300 �g/ml (three-fold dilutions) orin the presence of vehicle control (DMEM). As shown in Fig. 2b, SNPssignificantly inhibited PPRV replication at concentration as low as11.11 �g/ml, suggesting its antiviral efficacy against PPRV.

4 N. Khandelwal et al. / Virus Research 190 (2014) 1–7

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Fig. 2. In vitro efficacy of the SNPs against PPRV. (a) Determination of the cytoxicity (MTT assay): Three-fold serial dilutions of the SNPs or equivalent volume of the vehiclecontrol (DMEM) were incubated with the cultured Vero cells for 72 h and percentage of cell viability was measured by MTT assay. (b) In vitro anti-PPRV efficacy of the SNPs:Vero cells were infected with PPRV at MOI of 0.1 in the presence of various concentrations (three-fold serial dilutions) of the SNPs or vehicle control (DMEM). The virusparticles released in the supernatants were quantified by plaque assay. (c) Determination of the virucidal activity of the SNPs: Three-fold serial dilutions of the SNPs orequivalent volume of DMEM were mixed with the PPRV and incubated for 90 min at 37 ◦C after which the relative infectivity was determined by plaque assay. (d) Stabilityof the SNPs in cell culture medium: Aliquots of the SNPs were incubated at 37 ◦C for up to 7 days. At indicated time points, their anti-PPRV efficacy was evaluated in Veroc d usina

avctAut

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ells. Pair wise statistical comparisons to the vehicle control group were performend P < 0.001 respectively.

In order to determine whether the antiviral efficacy of the SNPsgainst PPRV is partially due to direct inactivation of the cell freeirion, we incubated infectious virions with the SNPs for 90 min atoncentrations ranging from 1.23 to 900 �g/ml or in presence ofhe vehicle control and then tested their infectivity on Vero cells.s shown in Fig. 2c, SNPs did not exhibit any virucidal effect evenp to 900 �g/ml suggesting anti-PPRV activity of the SNPs is due tohe inhibitory effect on virus replication in the target cells.

The stability of the SNPs in the cell culture medium wasvaluated by incubating them at 37 ◦C for up to 7 days and thenesting their anti-PPRV efficacy in Vero cells. As shown in Fig. 2d,o significant reduction in anti-PPRV efficacy was observed fromhe SNPs that were either freshly prepared or pre-incubated at7 ◦C over a period of 7 days suggesting SNPs are quite stable inhe cell culture medium.

.3. Effect of the SNPs on specific steps of PPRV life cycle

We have shown previously that the PPRV life cycle is 6–8 h inultured cells (Kumar et al., 2013). In order to study the stage(s) of

g Student’s t test. *, ** and *** represents statistical significance at P < 0.05, P < 0.01

the PPRV life cycle that can be affected by the SNPs, we performed atime-course assay, in which the SNPs were added at different timepre- and post-infection and the virus particles released into thesupernatant at 9 hpi were quantified by plaque assay. As shownin Fig. 3a, a significant reduction in the viral titers was observed,only when the SNPs were applied either 1 h prior to infection (pre-treatment) or at 1 hpi or 2 hpi suggesting that only the early step(s)of PPRV life cycle are affected by the SNPs. However, no signifi-cant inhibition in the viral titers was observed when the SNPs wereapplied at 4 hpi, 6 hpi and 8 hpi (Fig. 3a), suggesting later stages ofthe PPRV life cycle may not be affected by the SNPs.

In order to determine whether SNPs may block the PPRV attach-ment to the host cells, Vero cell monolayers were pre-incubatedwith SNPs for 1 h and then infected with PPRV at 4 ◦C for 2 h. Asshown in Fig. 3b, there was no significant difference in the viral

titers from the cell lysates prepared either from the SNPs-treatedor from the vehicle control-treated cells, suggesting SNPs do notinhibit PPRV attachment to the host cells. To further confirm thatthe SNPs do not affect virus attachment to the host cells, a HAI

N. Khandelwal et al. / Virus Research 190 (2014) 1–7 5

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Fig. 3. Step(s) of PPRV life cycle affected by SNPs. (a) Time-course assay: The confluent monolayers of Vero cells were infected, in triplicates, with the PPRV at MOI of 5,washed 6 times with PBS and fresh media having either 300 �g/ml of the SNPs or equivalent volume of the vehicle control were added at indicated times. The supernatantswere collected at 9 hpi and quantified in Vero cells by plaque assay. (b) Virus attachment assay: Vero cells were pre-incubated with either the SNPs (300 �g/ml) or equivalentvolume of the vehicle control for 1 h and then infected with the PPRV at 4 ◦C for 2 h. After the cells were washed 6 times with PBS, the cell lysates were prepared by rapidfreeze–thaw and the virus titers were determined by plaque assay. (c) Hemagglutination inhibition assay: Two-fold serial dilutions of heat inactivated anti-PPRV serum(starting from 1/8) and SNPs (starting concentration 300 �g/ml) were incubated with the PPRV (∼106 PFU/ml) for 1 h at 37 ◦C. Thereafter, 0.5% suspension of the RBCs wasapplied and the plates were incubated at 4 ◦C for about 2 h. The results were expressed as % hemagglutination inhibition. (d) Entry assay: The confluent monolayers ofVero cells were infected with the PPRV at MOI of 5 in SNPs-free medium for 1 h on ice to permit attachment. Thereafter, the cells were washed with chilled PBS to removeu re adda e medt ** repr

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nattached virus and the fresh medium containing either SNPs or vehicle control wegain with PBS to remove any extracellular virus and incubated with the cell culturitration of the progeny virus particles in the treated and untreated cells. *, ** and *

ssay was performed. As shown in Fig. 3c, PPRV attachment to thehicken RBCs was significantly inhibited in the presence of anti-PRV serum (HAI titer, 64) but not in the presence of the SNPs (HAIiter < 8) suggesting SNPs do not affect the PPRV attachment to theost cells.

To determine if pre-attached virus was able to enter the cellsn presence of the SNPs, a standard entry assay was performed.s shown in Fig. 3d, a significant reduction in the virus yield wasbserved in the SNPs-treated cells as compared to the vehicleontrol-treated cells, suggesting SNPs impair PPRV entry into theost cells.

PPRV virions are pleomorphic in shape which varies in size from50 to 700 nm (Boudin and Laurent-Vautiera (1967). The PPRV-NPs interaction was demonstrated by TEM analysis of the SNPs-reated PPRV. The SNPs were found to interact with the PPRV notnly at the level of virion surface (Fig. 4a) but also internalize tonteract with the virion core (Fig. 4b).

. Discussion

PPRV infection is economically so devastating that up to 100%f the animals (sheep and goats) on a farm/village may die withinew days. Though vaccine is considered as most effective mean for

ed. The entry was allowed to proceed at 37 ◦C for 1 h after which cells were washedia without any inhibitor. The cell culture supernatants were harvested at 9 hpi foresents statistical significance at P < 0.05, P < 0.01 and P < 0.001 respectively.

PPR control but cannot be used to provide instantaneous protec-tion during epidemics. Moreover, there are no antiviral medicationsavailable against PPR.

In this study the SNPs were biologically synthesized from sil-ver nitrate using aqueous extract of the Argimone maxicana whichhas been shown to exhibit a strong reducing property (Singh et al.,2010). The reduction of Ag+ ions and hence formation of the SNPswas confirmed by UV–vis spectroscopy. The XRD pattern revealedthe purity and characteristic shape (cubic and hexagonal) of theSNPs. The TEM analysis depicted their size ranging from 5 to 30 nm.The previous workers have also observed the SNPs size in the sim-ilar range viz.; 10 ± 2 nm (Li et al., 2007), 30–80 nm (Parasar et al.,2009) and 10–15 nm (Pareek et al., 2012). Further, the TEM analysisrevealed that the SNPs not only interact at the virion surface, butalso internalize to interact with the virion core as in case of HIV-1, Tacaribe virus, and HSV-1 (Baram-Pinto et al., 2009; Lu et al.,2008; Speshock et al., 2010). The precise nature of the reducingand capping agents present in the A. maxicana and other plantextracts, such as Capsicum annuum, Bougainvillea spectabilis that

have been used (Li et al., 2007; Parasar et al., 2009; Pareek et al.,2012; Singh et al., 2010) for biological synthesis of the SNPs is notknown. However, the size of the SNPs have been shown to affectantimicrobial/antiviral activity (Elechiguerra et al., 2005; Galdiero

6 N. Khandelwal et al. / Virus Research 190 (2014) 1–7

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ig. 4. The TEM micrograph of the SNPs-treated PPRV. (a) Interaction of the SNPs a

t al., 2011; Pal et al., 2007). The observed antiviral effect of theNPs is not simply because of silver ions present in the solution,ather it is due to the SNPs (Elechiguerra et al., 2005). The SNPs doot randomly attach to the virus particles; instead the interactionetween virus particles and SNPs is due to the size of the silveranoparticle because only SNPs within the range of 1–10 nm areble to bind to the virus (Elechiguerra et al., 2005).

In this study, a robust anti-PPRV efficacy of the SNPs wasbserved in vitro in Vero cells suggesting their potential as antivi-al therapeutics against PPR. Nevertheless, very recently, the SNPsave been found to inhibit replication of viruses such as hepatitis Birus (Lu et al., 2008), influenza virus (Xiang et al., 2011, 2013), her-es simplex virus (Gaikwad et al., 2013), Tacaribe virus (Speshockt al., 2010), vaccinia virus (Trefry and Wooley, 2013), adenovirusChen et al., 2013) and HIV-1 (Lara et al., 2010a,b, 2011). Thoughhe antiviral efficacy of the SNPs against several other viruses is stillo come but our study, together with these recent studies indicateshat the SNPs may have a broad spectrum antiviral effects.

The SNPs have been found to block different steps of virus repli-ation cycle. e.g. it has been found to inhibit vaccinia virus entryTrefry and Wooley, 2013), inhibit RNA synthesis by interferingith RNA-dependent RNA polymerase (L protein) and progeny

irus release in the Tacaribe virus (Speshock et al., 2010), damageber and capsid proteins of the adenovirus and hence inhibitingirus attachment to the host cell (Chen et al., 2013), bind enve-ope glycoprotein (gp120) of the HIV-1 to prevent internalizationf the virus particles into the cell (Lara et al., 2010a). The SNPs havelso been found to inhibit apoptosis of MDCK cells induced by thenfluenza H1N1 viruses (Xiang et al., 2011). We also attempted tonvestigate the stage(s) of the PPRV specifically affected by the SNPs.he time-course assay suggested that only early stages (before

hpi) of the PPRV life cycle are affected by the SNPs. The PPRVife cycle is of 6–8 h in cultured cells (Kumar et al., 2013). PPRVs an enveloped virus; it binds to host cell receptors (signaling

ymphocyte activation molecule; SLAM) via its surface envelopelycoprotein. The subsequent steps lead to fusion of the viral andhe host cell membrane and hence release of the virus particles intoytoplasm. The early steps of the PPRV life cycle, virus attachment to

irion surface. (b) Internalization of the SNPs and interaction with the virion core.

the host cell receptor, fusion of the viral and host cell membrane andrelease of the nucleocapsid in the cytoplasm are believed to occurduring first 2 h of infection. The SNPs in our study did not block thePPRV attachment to the host cells; rather it inhibited virus entryin the target cells. The SNPs were found to interact with the virionsurface as well with the virion core; however, this interaction didnot result in inactivation of the cell free virion. The SNPs-treatedPPRV was found to be equally competent (compared to untreatedPPRV) to form plaque on Vero cells suggesting SNPs do not blockthe receptor binding sites on PPRV envelope glycoprotein. Analo-gous to our study, a recent study on vaccinia virus indicates that theSNPs-treated vaccinia virus is capable of adsorbing but could notenter the cells (Trefry and Wooley, 2013). The virus particles thathad adsorbed to the cells in the presence of the SNPs may be infec-tious upon removal from the cells, indicating lack of direct virucidaleffect. It is a matter of further study to dissect the molecular mech-anism of interaction between the virion components, the SNPs andthe host cell factors that result in inhibition of the PPRV replicationin the target cells.

Apart from the size, the biological consequences of the SNPsalso depend on the surface structure, solubility, chemical compo-sition (capping agent), shape, and aggregation, which modify itsbiological interactions with the possibility of causing tissue injury(Galdiero et al., 2011). Therefore, safety of the SNPs is a major con-cern. Even if the SNPs inhibit viral infections, it would not haveany practical implications if there are adverse effects to humansor animals health. Several in vitro studies have demonstrated thecytotoxic effects of the metal nanoparticles, viz.; disturbances inmitochondrial functions, membrane leakage of lactate dehydroge-nase and abnormal cell morphologies (Braydich-Stolle et al., 2005;AshaRani et al., 2009; Kawata et al., 2009). However, it should beconsidered that the in vitro concentrations of the SNPs are oftenmuch higher than ones used in the in vivo experiments (Kim et al.,2008), therefore such exposures do not fully represent the in vivo

exposure. A commonly used strategy to reduce a possible toxic-ity is to use various capping agents to prevent the direct contactof the metal with the cells. Although the mechanism elucidatingnanomaterial toxicity is well understood, their long term effect on

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nimal/human health is still missing. Further in vivo research isarranted to setup safety guidelines for the SNPs and their futurese in the clinical setting.

Taken together, it was concluded that the SNPs impair the PPRVeplication at the level of virus entry into the target cells and thusas a potential as an antiviral therapeutic agent against PPR.

cknowledgements

Authors are grateful to Dr. D. Swarup (former Director, CIRG)or his administrative support to this study. Authors are thank-ul to Dr. K. Rajukumar, Senior Scientist, Central Instrumentationacility, High Security Animal Disease Laboratory, Indian Veteri-ary Research Institute, Bhopal, for the TEM analysis and Dr. M.ukherjee, Scientific Officer (Chemistry), Central Instrumentation

acility, Indian Institute of Science Education and Research, Bhopal,or XRD analysis.

eferences

shaRani, P.V., Low Kah Mun, G., Hande, M.P., Valiyaveettil, S., 2009. Cytotoxicityand genotoxicity of silver nanoparticles in human cells. ACS Nano 3, 279–290.

aram-Pinto, D., Shukla, S., Perkas, N., Gedanken, A., Sarid, R., 2009. Inhibition ofherpes simplex virus type 1 infection by silver nanoparticles capped with mer-captoethane sulfonate. Bioconjug. Chem. 20, 1497–1502.

aron, M.D., Parida, S., Oura, C.A., 2011. Peste des petits ruminants: a suitable can-didate for eradication? Vet. Rec. 169, 16–21.

oudin, P., Laurent-Vautiera, A., 1967. Note sur la structure du virus de la peste despetits ruminants. Rev. Elev. Méd. Vét. Pays Trop. 20, 383–386.

raydich-Stolle, L., Hussain, S., Schlager, J.J., Hofmann, M.C., 2005. In vitro cyto-toxicity of nanoparticles in mammalian germline stem cells. Toxicol. Sci. 88,412–419.

han, P., 2010. Exotic animal diseases bulletin. Peste des Petits Ruminants. Aust. Vet.J. 88, N20–N21.

harleston, B., Bankowski, B.M., Gubbins, S., Chase-Topping, M.E., Schley, D., Howey,R., Barnett, P.V., Gibson, D., Juleff, N.D., Woolhouse, M.E., 2011. Relationshipbetween clinical signs and transmission of an infectious disease and the impli-cations for control. Science 332, 726–729.

hen, N., Zheng, Y., Yin, J., Li, X., Zheng, C., 2013. Inhibitory effects of silver nanopar-ticles against adenovirus type 3 in vitro. J. Virol. Methods 193, 470–477.

lechiguerra, J.L., Burt, J.L., Morones, J.R., Camacho-Bragado, A., Gao, X., Lara,H.H., Yacaman, M.J., 2005. Interaction of silver nanoparticles with HIV-1. J.Nanobiotechnol. 3, 6.

aikwad, S., Ingle, A., Gade, A., Rai, M., Falanga, A., Incoronato, N., Russo, L., Galdiero,S., Galdiero, M., 2013. Antiviral activity of mycosynthesized silver nanoparti-cles against herpes simplex virus and human parainfluenza virus type 3. Int. J.Nanomed. 8, 4303–4314.

aldiero, S., Falanga, A., Vitiello, M., Cantisani, M., Marra, V., Galdiero, M., 2011. Silvernanoparticles as potential antiviral agents. Molecules 16, 8894–8918.

ibbs, E.P., Taylor, W.P., Lawman, M.J., Bryant, J., 1979. Classification of peste despetits ruminants virus as the fourth member of the genus Morbillivirus. Inter-virology 11, 268–274.

oris, N., Vandenbussche, F., De Clercq, K., 2008. Potential of antiviral therapy andprophylaxis for controlling RNA viral infections of livestock. Antiviral Res. 78,170–178.

ahn, W., 1999. Chemical aspects of the use of gold clusters in structural biology. J.Struct. Biol. 127, 106–112.

ones, J.C., Turpin, E.A., Bultmann, H., Brandt, C.R., Schultz-Cherry, S., 2006. Inhibi-tion of influenza virus infection by a novel antiviral peptide that targets viralattachment to cells. J. Virol. 80, 11960–11970.

search 190 (2014) 1–7 7

Kawata, K., Osawa, M., Okabe, S., 2009. In vitro toxicity of silver nanoparticles atnoncytotoxic doses to HepG2 human hepatoma cells. Environ. Sci. Technol. 43,6046–6051.

Kim, Y.S., Kim, J.S., Cho, H.S., Rha, D.S., Kim, J.M., Park, J.D., Choi, B.S., Lim, R., Chang,H.K., Chung, Y.H., 2008. Twenty-eight-day oral toxicity, genotoxicity, and gen-derrelated tissue distribution of silver nanoparticles in Sprague-Dawley rats.Inhal. Toxicol. 20, 575–583.

Kumar, N., Chaubey, K.K., Chaudhary, K., Singh, S.V., Sharma, D.K., Gupta, V.K., Mishra,A.K., Sharma, S., 2013. Isolation, identification and characterization of a Peste desPetits Ruminants virus from an outbreak in Nanakpur, India. J. Virol. Methods189, 388–392.

Kumar, N., Liang, Y., Parslow, T.G., 2011a. Receptor tyrosine kinase inhibitors blockmultiple steps of influenza a virus replication. J. Virol. 85, 2818–2827.

Kumar, N., Sharma, N.R., Ly, H., Parslow, T.G., Liang, Y., Parslow, T.G., 2011b. Receptortyrosine kinase inhibitors that block replication of influenza A and other viruses.Antimicrob. Agents Chemother. 55, 5553–5559.

Kumar, N., Xin, Z.T., Liang, Y., Ly, H., 2008. NF-kappaB signaling differentially regu-lates influenza virus RNA synthesis. J. Virol. 82, 9880–9889.

Kumar, N., Maherchandani, S., 2014. Targeting host cell factors for development ofantiviral therapeutics. Adv. Anim. Vet. Sci. 1S, 37–41.

Lara, H.H., Ayala-Nunez, N.V., Ixtepan-Turrent, L., Rodriguez-Padilla, C., 2010a. Modeof antiviral action of silver nanoparticles against HIV-1. J. Nanobiotechnol. 8, 1.

Lara, H.H., Ixtepan-Turrent, L., Garza-Trevino, E.N., Rodriguez-Padilla, C., 2010b.PVP-coated silver nanoparticles block the transmission of cell-free and cell-associated HIV-1 in human cervical culture. J. Nanobiotechnol. 8, 15.

Lara, H.H., Ixtepan-Turrent, L., Garza Trevino, E.N., Singh, D.K., 2011. Use of silvernanoparticles increased inhibition of cell-associated HIV-1 infection by neutral-izing antibodies developed against HIV-1 envelope proteins. J. Nanobiotechnol.9, 38.

Li, S., Shen, Y., Xie, A., Yu, X., Qiu, L., Zhang, L., Zhang, Q., 2007. Green synthesis ofsilver nanoparticles using Capsicum annuum L. extract. Green Chem. 9, 852–858.

Lu, L., Sun, R.W., Chen, R., Hui, C.K., Ho, C.M., Luk, J.M., Lau, G.K., Che, C.M., 2008. Silvernanoparticles inhibit hepatitis B virus replication. Antivir. Ther. 13, 253–262.

Mori, Y., Ono, T., Miyahira, Y., Quang, V., Nguyen, V.Q., Matsui, T., Ishihara, M.,2013. Antiviral activity of silver nanoparticle/chitosan composites against H1N1influenza A virus. Nanoscale Res. Lett. 8.

Pal, S., Tak, Y.K., Song, J.M., 2007. Does the antibacterial activity of silver nanopar-ticles depend on the shape of the nanoparticle? A study of the Gram-negativebacterium Escherichia coli. Appl. Environ. Microbiol. 73, 1712–1720.

Parasar, V., Parasar, R., Sharma, B., Pandey, A.C., 2009. Parthenium leaf extractmediated synthesis of silver nanoparticles: a novel approach towards weedutilization. Digest J. Nanomater. Biostruct. 4, 45–50.

Pareek, N., Dhaliwal, AbhijeetSingh, Malik, C.P., 2012. Biogenic synthesis of silvernanoparticles, using Bougainvillea spectabilis willd bract extract. Natl. Acad. Sci.Lett. 35, 383–388.

Rai, M., Yadav, A., Gade, A., 2009. Silver nanoparticles as a new generation of antimi-crobials. Biotechnol. Adv. 27, 76–83.

Rogers, J.V., Parkinson, C.V., Speshock, J.L., Hussain, S., 2008. A preliminary assess-ment of silver nanoparticle inhibition of monkeypox virus plaque formation.Nanoscale Res. Lett. 3, 129–133.

Singh, A., Jain, D., Upadhyay, M.K., Khandelwal, N., Verma, H.N., 2010. Green synthe-sis of silver nanoparticles using argemone Mexicana leaf extract and evaluationof their antimicrobial activities. Digest J. Nanomater. Biostruct. 2, 483–489.

Speshock, J.L., Murdock, R.C., Braydich-Stolle, L.K., Schrand, A.M., Hussain, S.M., 2010.Interaction of silver nanoparticles with Tacaribe virus. J. Nanobiotechnol. 8, 19.

Sun, L., Singh, A.K., Vig, K., Pillai, S.R., Singh, S.R., 2008. Silver nanoparticles inhibitreplication of respiratory syncytial virus. J. Biomed. Nanotechnol. 4, 149–158.

Trefry, J.C., Wooley, D.P., 2013. Silver nanoparticles inhibit vaccinia virus infectionby preventing viral entry through a macropinocytosis-dependent mechanism.J. Biomed. Nanotechnol., 1624–1635.

Xiang, D., Zheng, Y., Duan, W., Li, X., Yin, J., Shigdar, S., O’Connor, M.L., Marappan, M.,

Zhao, X., Miao, Y., Xiang, B., Zheng, C., 2013. Inhibition of A/Human/Hubei/3/2005(H3N2) influenza virus infection by silver nanoparticles in vitro and in vivo. Int.J. Nanomed. 8, 4103–4113.

Xiang, D.X., Chen, Q., Pang, L., Zheng, C.L., 2011. Inhibitory effects of silver nanopar-ticles on H1N1 influenza A virus in vitro. J. Virol. Methods 178, 137–142.