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Journal of Virological Methods 169 (2010) 232–238

Contents lists available at ScienceDirect

Journal of Virological Methods

journa l homepage: www.e lsev ier .com/ locate / jv i romet

hort communication

valuation of human intestinal epithelial differentiated cells (Caco-2) foreplication, plaque formation and isolation of avian influenza viruses

lam Jahangira,b, Sakchai Ruenpheta,c, Kazuya Harad, Dany Shohama, Nadia Sultanaa,asashi Okamuraa, Masayuki Nakamuraa, Kazuaki Takeharaa,c,∗

Laboratory of Zoonoses, School of Veterinary Medicine Kitasato University, JapanAnimal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka, BangladeshLaboratory of Animal Health, Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, JapanLaboratory of Microbiology, School of Allied Health Sciences, Kitasato University, Japan

rticle history:eceived 15 January 2010eceived in revised form 15 July 2010ccepted 22 July 2010vailable online 30 July 2010

eywords:

a b s t r a c t

Although various cultured cells are used for propagating influenza A viruses, the types of cells which cansupport replication of and plaque production by low pathogenic avian influenza (LPAI) viruses withoutsupplementary trypsin are limited. In this study, the infectivity and growth kinetics of as well as plaqueproduction by LPAI viruses in Caco-2 cells were investigated. The suitability of this cell line for virusisolation was examined and compared with virus isolation in embryonated chicken eggs. Generation ofCaco-2 mediated viral variants, if any, was assessed phenotypically and genotypically. It was found that

aco-2 cellsvian influenza viruseplicationlaque formationiral variants

Caco-2 cells can readily support continued replication of LPAI viruses without supplementary trypsin.Viruses replicate to high titer compared to embryonated chicken eggs, and more efficiently than in MDCKcells, without trypsin. Also, LPAI viruses produced plaques in Caco-2 cells. However, these cells werefound to be less sensitive than embryonated chicken eggs for virus isolation. Notably, no phenotypic andgenotypic changes of the viruses were observed during viral passages (at least up to 10th passage) inCaco-2 cells. These findings indicate that Caco-2 cells may provide an appropriate substrate for studying

and cultivating AIVs.

Wong and Kilbourne (1961) first described the accessibility ofuman and swine influenza viruses to heteroploid Chang conjunc-ival cells. Subsequently, several other cell lines or primary cellultures of mammalian and avian origins were reported as suit-ble for propagation of various strains of influenza A viruses (Gaushnd Smith, 1968; Govorkova et al., 1995; Hatano and Morita, 1967;ee et al., 2008; Sugimura et al., 2000; Sugiura and Kilbourne,965; Zaffuto et al., 2008). However, the inability of influenzairuses to produce plaques in the majority of the above cells lim-ts appreciably their usefulness (Hatano and Morita, 1967; Sugiurand Kilbourne, 1965). These reports deal mostly with human andwine influenza viruses, while only few refer to avian influenzairuses (AIVs). Since the discovery of enhanced growth stimulation

nd the effect of plaque production by trypsin supplementationn the culture medium (Tobita et al., 1975), Madin–Darby canineidney (MDCK) cells are used more frequently for AIVs studies,ncluding virus isolation from clinical and experimental specimens,

∗ Corresponding author at: Laboratory of Animal Health, Department of Vet-rinary Medicine, Tokyo University of Agriculture and Technology, Fuchu, Tokyo83-8509, Japan. Tel.: +81 42 367 5776; fax: +81 42 367 5776.

E-mail address: takehara@cc.tuat.ac.jp (K. Takehara).

166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2010.07.023

© 2010 Elsevier B.V. All rights reserved.

as well as samples from surveillance studies (Brown et al., 1993;Gourreau et al., 1994; Sugimura et al., 2000). While MDCK cellsrepresent a mammalian substrate used commonly for studyingboth mammalian influenza viruses and AIVs, the human origi-nated, Caco-2 cells have been shown to be useful for mammalianinfluenza viruses (Chiapponi et al., 2010; Yoshino et al., 1998;Zhirnov and Klenk, 2003). Recently, it was reported that highlypathogenic avian influenza (HPAI) H5N1 virus can replicate effi-ciently in Caco-2 cells (Li et al., 2009). However, there is lack ofstudy on Caco-2 cells with low pathogenic avian influenza (LPAI)viruses. Zhirnov and his colleagues accomplished several studieswith mammalian influenza viruses, and documented that (i) Caco-2 cells contain both �2-3 linked sialic acid receptors (specific forAIVs) and �2–6-linked sialic acid receptors (specific for humaninfluenza viruses); (ii) human influenza viruses can continue mul-ticycle replication in Caco-2 cells without supplementary trypsin(Zhirnov and Klenk, 2003; Zhirnov et al., 2003, 2007; Zhirnov andSyrtzev, 2009).

Yet, to this day, the number of reported cell cultures suitablefor replication of and plaque production by AIVs is limited. Properplaque assay systems for AIVs are desirable for adequate quanti-tative profiling, and might be of great value for genetic studies.Considering the findings obtained by Zhirnov et al. (2007), par-

A. Jahangir et al. / Journal of Virological Methods 169 (2010) 232–238 233

Table 1Efficiencies of low pathogenic avian influenza (LPAI) virus’ replication and plaque formation in Caco-2 cellsa.

Virusb EID50/ml (log10) PFU/ml (log10) on Caco-2c TCID50/ml (log10)

Caco-2 MDCK

A/Np/Aomori/422/07 H1N1 9.0 6.2/7.2 8.1 ± 1.4/8.2 ± 0.9d 3.7 ± 0.4/7.1 ± 0.5A/Np/Miyagi/1472/08 H1N1 9.0 5.4/6.3 6.6 ± 0.2/8.5 ± 0.5 5.0 ± 0.7/6.4 ± 0.4A/Np/Aomori/1130/08 H1N3 8.5 5.4/6.3 7.4 ± 1.3/7.8 ± 0.9 3.8 ± 0.6/6.5 ± 0A/Bg/Aichi/1/77 H3N8 7.5 3.4/5.7 2.5 ± 0/5.4 ± 0.2 5.2 ± 0.6/6.2 ± 0.1A/Np/Miyagi/1733/07 H4N6 9.5 5.3/5.7 6.2 ± 0.5/6.5 ± 1.8 3.5 ± 0/5.6 ± 0.2A/Np/Akita/714/06 H5N2 8.0 4.3/5.3 7.1 ± 0.3/8.0 ± 0.5 5.9 ± 0.2/7.0 ± 1.4A/Np/Aomori/385/08 H5N3 9.0 4.7/5.6 6.8 ± 0.3/7.9 ± 0.5 5.1 ± 0.1/6.6 ± 0.8A/Np/Aomori/1192/08 H5N9 6.5 5.3/6.3 5.7 ± 0.8/7.8 ± 1.2 4.4 ± 1.2/6.4 ± 0.5A/Np/Miyagi/258/05 H6N2 7.5 5.2/6.0 6.8 ± 1.1/8.4 ± 0.6 6.0 ± 0.4/6.3 ± 0.7A/Np/Miyagi/411/06 H6N8 7.5 5.4/6.0 6.6 ± 0.2/7.1 ± 0.9 3.6 ± 0.8/6.7 ± 0.3A/Np/Akita/762/06 H6N1 8.5 6.5/7.2 6.8 ± 0.5/7.7 ± 0.8 4.3 ± 1.4/6.7 ± 0.2A/Np/Aomori/395/04 H7N1 8.5 6.2/6.2 7.8 ± 0.5/7.6 ± 0.8 6.4 ± 0.2/6.4 ± 0.5A/Np/Aomori/372/08 H7N7 7.5 5.7/5.9 6.6 ± 1.0/6.7 ± 0.2 4.3 ± 0.9/5.9 ± 0.3A/Np/Akita/1366/08 H7N7 –e 5.3/6.0 6.1 ± .1/6.6 ± 0.5 4.1 ± 0.5/6.8 ± 0.9A/Np/Aomori/873/06 H10N1 8.0 5.3/6.4 6.3 ± 0/6.8 ± 0.6 5.5 ± 0/6.4 ± 1.3A/Np/Aomori/926/08 H10N9 – 5.4/6.3 7.1 ± 0.6/7.9 ± 1.2 3.5 ± 0/6.9 ± 0.3A/Np/Akita/131/07 H11N9 8.0 4.5/6.1 6.1 ± 0.3/7.7 ± 0.1 4.1 ± 0.5/5.4 ± 0.6

a Viruses were initially isolated in embryonated chicken eggs from fecal materials and virus in allantoic fluid was stored at −80 ◦C. For this experiment, viruses werepassaged again to prepare working virus. Serial 10-fold dilutions of each of the working viruses were made with serum free medium and employed in embryonated chickeneggs and cell inoculation and plaque assay.

b Abbreviations: Np: Northern pintail; Bg: Budgerigar.

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c Without trypsin/with trypsin (2 �g/ml).d Mean ± standard deviation.e Not tested.

icularly in that Caco-2 cell contain receptor for AIVs, it is hereresumed that this cell line may be suitable for AIVs studies, pos-ibly advantageously. In addition, being an immortalized cell line,ne potential advantage could be lasting exploitability. This reportresents, for the first time, comprehensive evidence and implica-ions pertaining to infectivity of and plaque formation by variousPAI viruses in Caco-2 cells. Furthermore, the extent of applica-ility of this cell line for isolation of AIVs from fecal samples andxperimental specimens was shown.

A total of 17 LPAI viruses were used in this study (Table 1).he viruses were taken from a repository of the Zoonoses Lab-ratory at Kitasato University. All of these viruses were isolatedn embryonated chicken eggs from fecal materials of migratory,pparently healthy northern pintail (Anas acuta) ducks in JapanJahangir et al., 2008, 2009a), except for A/budgerigar/1/77 H3N81/77 H3N8), which was purchased from Japan Veterinary Productsssociation. Working stocks of viruses were prepared by passage

n embryonated chicken eggs, and allantoic fluid was harvestedt 3 days post inoculation (dpi), and stored at −80 ◦C for furtherse.

Caco-2 cells were maintained in Dulbeco’s Modified Eagleedium (DMEM, Nissui Pharmaceutical Co. Ltd., Tokyo, Japan), sup-

lemented with penicillin 100 units/ml, streptomycin 100 �g/ml,mphotericin B 0.5 �g/ml, 4 mM l-glutamine, and heat-inactivated0% (v/v) fetal calf serum (FCS), and were incubated at humid-

fied environment at 37 ◦C in CO2 (5%) incubator. Initially theuitability of Caco-2 cells for replication of AIV was examinedsing an AIV strain, A/Northern pintail/Akita/714/06 H5N2 (714/065N2). Monolayer of Caco-2 cells was inoculated with differentoncentrations of 714/06 H5N2 in a 96-well tissue culture plateCellstar, Greiner Bio-one Co., Tokyo, Japan). Then the plate wasncubated at 37 ◦C and cytopathic effect (CPE) was observed for 3ays. Virus presence was determined by haemagglutination testsing 0.5% suspension of chicken red blood cells (RBC). Culture

uid was tested by haemagglutination inhibition (HI) test withnti-H5 serum (Alexander, 2004). Besides, reverse transcriptionolymerase chain reaction (RT-PCR) was done with culture fluid toonfirm the presence of H5N2 virus, following previously describedethods (Hoffmann et al., 2001; Lee et al., 2001).

The tolerability of Caco-2 cells to supplementary trypsin wasdetected by a dose response assay. Monolayer of Caco-2 cellsin a 96-well plate was added with medium containing 0, 1,2, 4, 8, and 10 �g/ml (final concentration) supplementary l-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treatedbovine pancreatic trypsin (Sigma, USA), and incubated at 37 ◦C.Integrity of the cells was observed for 3 days. The effect of sup-plementary trypsin on virus titer yield was also measured by adose response assay. The 714/06 H5N2 was used as a test virus.Monolayer of Caco-2 cells was replaced with medium (100 �l/well)containing 0, 2, 4, 8, and 16 �g/ml trypsin. Then, 100 �l of serially10-fold diluted virus was added per well that makes trypsin con-centration (final) 0, 1, 2, 4, and 8 �g/ml. The plate was incubated at37 ◦C for 3 days. Virus presence was determined by haemaggluti-nation test. The fifty percent tissue culture infectious dose (TCID50)was resolved by the Spearman–Kaerber method (Villegas, 2008).

Titrations of working viruses (Table 1) were performed in 10-day old embryonated chicken eggs by inoculating serial 10-folddilutions of virus into the allantoic cavity, and incubation at 37 ◦Cfor 3 days. At the end of incubation, allantoic fluid was harvestedseparately, and tested for virus by haemagglutination test. The fiftypercent embryo infectious dose (EID50) was determined as men-tioned above. Besides, viruses were also titrated in Caco-2 andMDCK cells, in the presence or absence of supplementary trypsin(2 �g/ml). Titrations were done using standard protocol. Briefly, a96-well tissue culture plate was seeded with ∼7 × 104 cells/well.At 100% confluent monolayer cells were washed thrice with PBS,and finally replaced with serum free medium 100 �l/well, with orwithout trypsin. A 10-fold serial dilution of each virus (10−3–10−10)was prepared with the medium. After infection of cells with dilutedvirus (100 �l/well and 4 wells for each dilution), the plate was incu-bated at 37 ◦C for 3 days. Wells defined as negative control wereinoculated with serum free medium. Presence of virus in the culturefluid was determined by haemagglutination test at 3 dpi, and the

virus titer, namely median tissue culture infection dose (TCID50),was determined (Villegas, 2008). The titration was carried out intriplicate.

Plaque assay was performed in Caco-2 and MDCK cells in thepresence or absence of supplementary trypsin. Monolayer of Caco-

2 logica

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ttiBag7bTt2sfltH

34 A. Jahangir et al. / Journal of Viro

cells in a 60 mm tissue culture Petridish (herein dish(es)) wasnoculated with 100 �l/dish of serial 10-fold diluted virus andncubated at 37 ◦C for 60 min. First overlay medium, consistingf 0.8% type II agarose (Sigma) in maintenance medium with 1%CS, was added after adsorption and washing of cells. Two daysater, the second overlay medium, containing 1% neutral red, wasdded to facilitate plaque counting. The plaque forming units (PFU)ere resolved, and size of plaques was measured at 3 dpi. Trypsin,�g/ml as final concentration, was added to the first overlayedium instead of 1% FCS, when plaque assay was performed in

he presence of trypsin.Adsorption kinetics of LPAI viruses was assessed in Caco-2 cells.

or this purpose, monolayer of cells in a 60 mm dish was washedhrice with PBS, and was inoculated with viruses at multiplicityf infection (MOI) of 10 TCID50/cell. At 15, 30, 45, and 60 min postxposure, the inoculum was removed, and the dish was washedhrice with PBS to remove residual viruses. Cells were dislodgedy polystyrene rubber cork and resuspended in 1 ml of serum freeedium. Sonication of cells was performed on ice by three 10 s

ursts, with 30 s interval, using a Handy Sonic UR-20P (Tomy Seikoo. Ltd. Tokyo, Japan). The obtained suspension was centrifuged at2,000 × g for 3 min at 4 ◦C, followed by titration in Caco-2 cells asescribed above.

For growth kinetics, monolayer of Caco-2 cells in a 60 mm dishas washed, and infected with virus in allantoic fluid at MOI of

.1, 0.01 and 0.001 TCID50/cell. After tilting, the dish was incubatedt 37 ◦C for 60 min. To facilitate better adsorption, tilting was per-ormed every 15 min during incubation, and the inoculum was thenemoved. The monolayer was washed thrice, added with serum freeedium, and incubated at 37 ◦C. Culture supernatants of 1 ml were

ollected for 4 days at 24 h intervals, centrifuged at 12,000 × g formin, and stored at −80 ◦C until tested. After each collection, 1 ml of

erum free medium was added to dishes. For one-step replication,ells were infected with 10 TCID50/cell, and samples were collectedt 2 h intervals for 0–16 h. The collected samples were titrated inaco-2 cells as mentioned above.

The effect of serum on viral replication was resolved by methodescribed earlier (Zhirnov et al., 2002). Briefly, a monolayer of cells

n a 6-well plate was washed thrice with PBS (pH 7.2), and infectedith A/Northern pintail/Aomori/422/07 H1N1 (422/07 H1N1), with

n MOI of 0.001 TCID50/cell. One hour post exposure to virus at7 ◦C, the monolayer was washed as mentioned above. Finally, cellsere incubated in medium with 0%, 0.5%, 2.0%, 8.0%, and 12% heat-

nactivated calf serum (CS). MDCK cells were incubated with DMEMontaining trypsin (2 �g/ml), and an amount of serum as listedbove. All cells were maintained at 37 ◦C in a 5% CO2 atmosphereor 3 days. One milliliter of culture supernatant was collected dailynd replaced with equal volume of medium containing percent-ge of serum mentioned above. Samples were titrated as describedbove.

To assess the suitability of Caco-2 cells for AIV isolation, aotal of 994 fresh composite fecal materials of northern pin-ail ducks were tested both in embryonated chicken eggs andn Caco-2 cells, as described previously (Jahangir et al., 2009b).riefly, 25% suspension of fecal sample was made with 100Xntibiotics (penicillin 10,000 units/ml, streptomycin 10,000 �g/ml,entamycin 5000 �g/ml, and amphotericin B 50 �g/ml) in PBS (pH.2). After keeping overnight at 4 ◦C, the supernatant was separatedy centrifugation at 12,000 × g for 3 min and regarded as sample.wo hundred microliter of samples were inoculated into each ofhe two 10-day old embryonated chicken eggs and two wells of

4 well tissue culture plate having monolayer of Caco-2 cells anderum free medium with trypsin 2 (�g/ml). On 3 dpi, the allantoicuid and cell culture supernatant were assayed by haemagglutina-ion test. Virus was identified from haemagglutinating samples byI and RT-PCR as mentioned above. Samples showing no haemag-

l Methods 169 (2010) 232–238

glutinating activity in the first passage were pooled (5 samplespooled) and tested further in the embryonated chicken eggs andCaco-2 cells before considered as negative. Virus isolation was alsoattempted from oropharyngeal swabs, following intra-ocular andintra-nasal, experimental infections of chickens, with A/Northernpintail/Aomori/395/04 H7N1 (395/04 H7N1) virus. Oropharyngealswabs were collected from 1 to 5 dpi and put into 1 ml of trans-fer medium containing equal amounts of 2X brain heart infusionand 100X antibiotics, and kept at 4 ◦C overnight. Supernatant wascollected and used for virus isolation in embryonated chicken eggsand in Caco-2 cells, as mentioned above.

To explore whether replication of AIVs in Caco-2 cells selectshost cell mediated variants, phenotypic, antigenic, and geneticanalyses of the virus isolated in embryonated chicken eggs and pas-saged in Caco-2 cells were conducted. The 395/04 H7N1 (embryopassage3-E3) virus was serially passaged in Caco-2 cells (at presentup to 10 serial passages). For serial passages, monolayer of cells ina 60 mm dish was inoculated with MOI of 0.001 TCID50/cell andincubated at 37 ◦C for 3 days. Upon incubation, supernatant washarvested and virus presence was determined by haemagglutina-tion test. The obtained virus was designated as 395/04 H7N1E3Caco-2-1 (H7N1E3 Caco-2-1). The H7N1E3 Caco-2-1 was dilutedto set appropriate MOI and inoculated again into Caco-2 cells asmentioned, repeatedly up to 10 serial passages. Phenotypic andantigenic analyses were done by profiling the viral haemagglutinat-ing property and HI activity, respectively. Genotypic stability wasdetermined by sequencing the haemagglutinin (HA) gene, whichis basically liable to undergo changes. Full-length HA gene wasamplified as described by Hoffmann et al. (2001). PCR product waspurified from gel using QIAquick Gel Extraction Kit (Qiagen, USA)according to manufacturer’s instructions. Full-length protein cod-ing region of HA gene was sequenced by cycle sequencing withinfluenza virus segment specific primers (Hoffmann et al., 2001),and subsequently by designed sequence-specific primers. Cyclesequencing was performed with a Big Dye Terminator kit (AppliedBiosystems, Foster City, CA), and run on an ABI 310 (Applied Biosys-tems), edited and assembled with GENETYX-Mac (version 10.0;Software Development Corp., Tokyo, Japan).

Series of studies aiming to explore the applicability of Caco-2cells in relation to LPAI viruses were performed. As a starting point,it was checked whether LPAI virus could grow in Caco-2 cells andfound that LPAI 714/06 H5N2 can replicate and produce round typeCPE in Caco-2 cells. The 714/06 H5N2 virus presence in the culturesupernatant was further confirmed by HI test and RT-PCR (datanot shown). Besides, NS1 synthesis in LPAI virus infected Caco-2cells was confirmed by Western blotting, using monoclonal anti-body to NS1 developed in the lab (personal communication). It iswell established that a protein not incorporated into the virion,but synthesized in AIV-infected cells, is NS1. Therefore, presence ofCPE and NS1 in virus infected Caco-2 cell along with virus detectionresults strongly corroborates the active replication of LPAI virus inthis cell system. In another study, the tolerability of Caco-2 cellsto supplementary trypsin and effect of supplementary trypsin onvirus titer yield was detected by a dose response assay. Amongthe tested doses (0, 1, 2, 4, 8, and 10 �g/ml trypsin as final con-centration), trypsin concentration of 4 �g/ml was found to be theoptimal concentration that Caco-2 cells can withstand without los-ing cell integrity. However, 2 �g/ml trypsin as final concentrationwas found to enhance 1 log10 higher titer than the other doses(1, 2, and 4 �g/ml) used for this study (data not shown). Conse-quently, 2 �g/ml trypsin as final concentration was chosen and used

throughout the quantitation assay.

Comparative quantitations of different subtypes of LPAI viruseswere performed in Caco-2 and MDCK cells and in embryonatedchicken eggs. Basically, the tested viruses replicated well in Caco-2 cells, comparable to titers obtained in embryonated chicken

A. Jahangir et al. / Journal of Virological Methods 169 (2010) 232–238 235

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observed. Significant accumulations of viruses in the culture fluidswere observed at 2 dpi for all viruses, except for A/Northern pin-tail/Akita/131/07 H11N9, where peak titer was achieved at 3 dpi.However, reduction of virus titer was evident at 3 dpi for the

Fig. 1. Plaque production by LPAI viruses in Caco-2 cells. (a) uninfected

ggs. Titer yield was found lower markedly in MDCK cellsithout supplementary trypsin. However, addition of trypsin inDCK cell culture medium was found to increase the virus titer

ield, comparable to both Caco-2 and embryonated chicken eggsTable 1). The mean titer variations in the absence and pres-nce of supplementary trypsin were approximately 1 log10 forhe majority of viruses in Caco-2 cells. Nevertheless, higher titers1.9–2.9 log10) were observed with supplementary trypsin for/Northern pintail/Miyagi/1472/08 H1N1 (1.9 log10), 1/77 H3N8

2.9 log10), and A/Northern pintail/Aomori/1192/08 H5N9 (1192/085N9) (2.1 log10) viruses. Viral titers in Caco-2 cells were foundomparable to titers obtained in embryonated chicken eggsTable 1). However, without supplementary trypsin, in about fiftyercent of the cases the mean TCID50 of each of the viruses wasound to be 0.7–1.1 log10 lower than EID50. Notably, when supple-

entary trypsin was added in Caco-2 cells culture medium, theifferences between EID50 and TCID50 became narrower (Table 1).he findings of increased TCID50 in Caco-2 cells in the presence ofupplementary trypsin comply with the findings of Zhirnov andlenk (2003) and Zhirnov et al. (2003). They reported that about0–20% of the viruses released from infected Caco-2 cells possesA0. It is assumed that supplementary trypsin cleaves the HA0 of

eleased viruses, thereby enabling them to then infect new cellsnd thus increase the yield of virus. Comparative quantitation stud-es of LPAI viruses in embryonated chicken eggs and in Caco-2ells revealed that both systems were considerably sensitive. Whenompared to MDCK cells, Caco-2 cells were found more sensitivehan MDCK cells in the absence of supplementary trypsin in MDCKell culture medium.

Plaque test was done with or without extraneous trypsin, usingype II agarose at a concentration of 0.8 g/ml. All the tested LPAIiruses produced plaques on Caco-2 cells (Table 1). The number ofFU was found to vary for different viruses, and the plaque titer wasncreased considerably when trypsin was added to the first overlay

edium. No plaque was found in control dish (Fig. 1a). The diame-er of the plaques, as measured at 3 dpi, ranged from 0.5 to 3.5 mmFig. 1b). However, obscure plaques difficult to count and mea-ure were also found in the present study. Although the numberf plaques increased for a given virus with the addition of trypsin,otable effect on plaque size was not observed. The increased num-er of plaques in the presence of supplementary trypsin might beue to cleavage activation of HA0 in uncleaved viruses releasedrom infected Caco-2 cells (Zhirnov and Klenk, 2003; Zhirnov et al.,

003).

Attachment is essential for a given virus to then enter the hostell. In the case of AIVs, this function is arbitrated by HA glyco-rotein with the related receptors present on host cell surface.epresentatively, the adsorption rates of 422/07 H1N1 and 395/04

ol and (b) plaques produced by 422/07 H1N1, as documented on 3 dpi.

H7N1 viruses were assessed after exposure of cells to virus for vari-ous periods. From the results of these experiments, it was apparentthat the tested viruses were adsorbed promptly to the Caco-2 cells.The adsorption rate peaked by 30 min of exposure (Fig. 2), and thuspersisted for up to 60 min for both viruses. The adsorption rate ofthe two tested viruses did not differ markedly. The characteris-tics of influenza A virus replication in cells are established for long(Gaush and Smith, 1968; Tobita et al., 1975). It was found that AIVswere adsorbed to Caco-2 cells quickly, comparable to MDCK (Gaushand Smith, 1968), and faster than in G2 cells and primary mon-key kidney cells (Choppin, 1962; Hatano and Morita, 1967). In part,these findings could reflect the diversity of both biological systems(including types of cells, and viruses) as well as methods that havebeen employed.

AIVs representing various HA subtypes (H1, H5, H7, H10, andH11) with different NA subtype combinations were used in thepresent study to investigate the kinetics of virus replication with-out extraneous trypsin. Monolayer of cells was infected withviruses at different MOI, and culture supernatant was collectedat the indicated times post exposure of cells to virus. At MOI of0.001 TCID50/cell the tested viruses replicated markedly in Caco-2cells, with high titer showing a range of 106.5–7.0 TCID50/ml (Fig. 3).Notable differences in peak titer of different viruses were not

Fig. 2. Adsorption of avian influenza viruses to Caco-2 cells infected with10 TCID50/cell. At each indicated time point, cells were washed thrice with PBS, anddislodged by polystyrene rubber cork. Cells were resuspended in 1 ml of serum freemedium and sonicated on ice. After centrifugation at 12,000 × g for 3 min, sampleswere titrated in Caco-2 cells in the presence of trypsin (2 �g/ml).

236 A. Jahangir et al. / Journal of Virological Methods 169 (2010) 232–238

Table 2Effect of serum on replication of LPAI H1N1 virus in Caco-2 and MDCK cells at 3 days post inoculationa.

Cell line Virus yield (log10 TCID50/ml) at CS concentrations of:

0% 0.5% 2% 4% 8% 12%

Caco-2 7.6 ± 0.8b 6.67 ± 0.38 7.42 ± 0.14 7.25 ± 0.25 7.5 ± 0.25 6.92 ± 0.52MDCK 6.4 ± 0.5 6.4 ± 0.4 3.5 ± 0.4 1.5 ± 0.3 1.5 ± 0 1.5 ± 0

7 H1Nw millilm e cul

mosmCcr(ppclwiwo3g(mcnwCHcbwaa

F2wmew

a Monolayers of Caco-2 and MDCK cells in 6-well plates were infected with 422/0ere incubated in medium containing different concentrations of calf serum. Oneedium containing percentage of serum mentioned above. Trypsin was added to thb Mean ± standard deviation.

ajority of viruses, and continued to day 4. Similar results werebtained when MOI was increased to 0.01–0.1 TCID50/cell (data nothown). These data indicate that Caco-2 cells can readily supportulticycle replication LPAI viruses without supplementary trypsin.

leavage activation of HA protein, which is synthesized as pre-ursor HA0, into HA1 and HA2 units is essential for multicycleeplication of AIVs, thus constituting a prerequisite for virulenceKlenk and Garten, 1994; Steinhauer, 1999). At the same time,ersisting multicycle replication may take place sub-clinically inermissive but tolerant host species. To determine whether HA0leavage of LPAI viruses cultured in Caco-2 cells occurs intracellu-arly, effect of serum on the replication of LPAI virus in Caco-2 cells

as examined. AIV-infected MDCK cells with exogenous trypsinn their culture fluid were used as control. At 1 dpi viral growth

as not detected in the sample supplemented with 12% CS, whilebserved at other concentrations (data not shown). However, bydpi the virus could replicate to an extent comparable to controlroup (0% CS) even in the presence of high concentration of serum12%) in Caco-2 cells (Table 2). By contrast, in MDCK cells supple-

ented with trypsin, the virus titer was found low when serumoncentrations were 2% or greater. Thus, virus activation by exoge-ous trypsin, which is necessary for virus growth in MDCK cells,as effectively inhibited in serum-containing medium, but not inaco-2 cells. From this study it is clear that cleavage activation ofA occurred inside the Caco-2 cells, as evidenced by virus repli-

ation in the presence of CS. However, replication was found toe delayed (at 1 dpi no viral growth was found in case of samplesith 12% CS). It is known that serum contains antiproteases, such

s alpha2-macroglobulin, alpha1-antitrypsin, alpha2-antiplasmin,nd antithrombin III (Travis and Salvesen, 1983), which may com-

ig. 3. Multicycle replication of low pathogenic avian influenza viruses in Caco-cells infected with multiplicity of infection (MOI) 0.001 TCID50/cell. Cells wereashed thrice after 1 h of adsorption and incubated at 37 ◦C with 3 ml of serum freeedium. At interval, 1 ml of culture supernatant was removed and replaced with

qual volume of the same medium. Samples were stored at −80 ◦C until virus titeras resolved.

1 virus at a multiplicity of infection (MOI) of 0.001 TCID50/cell in 100 �l. Then, cellsiter of culture supernatant was collected daily and replaced with equal volume ofture medium of MDCK cells at final concentration of 2 �g/ml.

pete for proteases on the cell surface, or in the extracellular fluid ofinfluenza virus infected Caco-2 cells. These findings are in compli-ance with a previous report (Zhirnov and Klenk, 2003; Zhirnov etal., 2003). In another study, Iki et al. (2005) reported that in Caco-2cells influenza virus replicates more rapidly at lower fetal bovineserum concentrations (0% or 2%) than at higher concentrations (10%or 20%) during an early stage of infection. Delayed replication ofLPAI virus in Caco-2 cells in the presence of high concentrations ofCS (12%) also supports the above findings.

To define one cycle replication of AIVs, Caco-2 cells wereinfected with H1N1 and H7N1 viruses at MOI of 10 TCID50/cell, sep-arately. One hour post adsorption at 37 ◦C the cells were washedand incubated with serum free medium. At indicated time points,1 ml of supernatant was taken and replaced with equal volume ofthe same medium. Resulting growth curves of viruses in Caco-2cells (Fig. 4) indicate that the tested viruses can complete one cyclereplication within 10–12 h.

Applicability of Caco-2 cells for primary isolation of AIVs wasassessed. As a part of a surveillance study, fecal materials of migra-tory northern pintail ducks were collected, and portion of thesamples (n = 994) were analyzed by both embryonated chicken eggsand Caco-2 cells for haemagglutinating virus isolation and subse-quent identification. Of these samples, 12 (1.21%) and 2 (0.20%)were found positive by embryonated chicken eggs and Caco-2 cells,respectively. The two latter samples were also found positive inembryonated chicken eggs. Out of 120 oropharyngeal swabs testedboth in embryonated chicken eggs and in Caco-2 cells, 11 sampleswere found positive by both systems, while 53 samples were foundpositive in embryonated chicken eggs only. In contrast, none of thesamples were regarded as positive only in Caco-2 cells. The levelof agreement between the results obtained by both systems wasfound to range from 53.8% to 98.9%. However, the virus isolation

rate was lower in Caco-2 cells, compared to embryonated chickeneggs. These findings differ from findings of Zhirnov et al. (2007),who showed that Caco-2 cells are sensitive 100 times higher thanMDCK cells and embryonated chicken eggs, for isolation of human

Fig. 4. One-step replication of LPAI viruses in Caco-2 cells. Monolayer of cells wasinfected with multiplicity of infection (MOI) 10 TCID50/cell. At indicated time points,samples were collected and replaced by equal volume of serum free medium. Virustiter was determined in the presence of 2 �g/ml.

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A. Jahangir et al. / Journal of Viro

nfluenza viruses from respiratory tract origin clinical specimens.his, in part, reflects the higher affinity of human influenza viruseso human originated cells, although they contain �2-3 linked sialiccid receptors specific for AIVs as well. In a recent study, Chiapponit al. (2010) investigated swine influenza virus isolation, usingmbryonated chicken eggs, Caco-2 cells, and MDCK cells from theCR positive sample. They found that the Caco-2 cell line is moreensitive than the MDCK cells and embryonated chicken eggs forhe isolation of H1N1 and H1N2 subtypes. However, they alsoeported that H3N2 virus was isolated more readily in embryonatedhicken eggs than in cultured cells. Swine strains are often close touman strains, and the observed variance may apparently stem

rom viral genomes containing more avian-related genes within3N2 than H1N1 and H1N2 (as is the case with human H1N1 and1N2 strains). The possible presence of biological inhibitory sub-

tances in the avian originated fecal materials and oropharyngealwabs used in the present study could affect the extent of sensitivityf Caco-2 cells. In addition, the methods applied here to isolate virusight have some effect. This initial study established, though, that

aco-2 cells constitute in some senses an advantageous permissiveissue culture for AIV. To be more conclusive regarding the appli-ability of Caco-2 cells for virus isolation and propagation, moreamples from naturally and experimentally infected avian speciesave to be tested, possibly with somewhat altered methods.

Phenotypic, antigenic and genetic studies were conducted toxplore whether replication of AIVs in Caco-2 cells selects hostell mediated variants. Phenotypic analysis was done by profilinghe haemagglutinating activity of the virus. The haemagglutinatingctivity of the viruses did not change during several passages (cur-ently 10 passages) in Caco-2 cells at MOI of 0.001 TCID50/cell, whenompared to the original virus isolated in embryonated chickenggs. Furthermore, marked changes – either increase or decrease inlaque number and in plaque size produced by original and Caco-2ells-passaged viruses – were not observed. Likewise, the antigenicrofile generated by HI test using reference polyclonal chicken anti-era to H7 virus, showed no variance between original and Caco-2ells-passaged viruses. Sequence analysis of the HA gene revealedhat the virus retained its amino acid pattern 217QSG (H7 number-ng) that binds preferentially to �2-3 linked sialic acid receptorspecific for avian viruses. The amino acid motif of the HA0 cleavageite was found the same IPKGR*GLF (* cleavage site) in both originalnd Caco-2 cell passaged viruses. Isolation of influenza A viruses inmbryonated chicken eggs or cell culture is critical for epidemi-logical investigations and for production of vaccines. However,t is known that host cell has significant effect on viral antigenictability and might generate host cell mediated variants. A similarhenomenon was reported by Zhirnov et al. (2007). They observehat clinical viruses isolated in Caco-2 cells, when further passagedn this cell (at least 20 passages), retain the original human RBCgglutination property (human phenotype) (not chicken RBC). Inontrast, viral passages in MDCK cells generate avian phenotypeagglutination of chicken but not human RBC) variants within 3–5assages. Changes neither in phenotypic or antigenic properties,or in receptor specificity of the virus passaged in Caco-2 cells (at

east 10 passages) were observed in the present study. This propertyf Caco-2 cells represents a further advantage in relation to AIV, andlaborates the previous observation. Recently, Zhirnov et al. (2009)ompared the primary structures of several genes of three H3N2iruses isolated from clinical samples to viruses passaged in Caco-2nd MDCK cells. They did not find any mutation in the HA recep-or binding residues specific to cell line. However, they observed

hanges in other genes, including NA. In the present study, only theA gene of H7N1 virus was sequenced and compared. The receptorinding site and HA cleavage site were found identical in originalnd Caco-2 and MDCK cells-passaged viruses. Besides, seven gly-osilation sites (motif Asn-X-Thr/Ser) were found to be stable in

l Methods 169 (2010) 232–238 237

original and passaged viruses. These findings were in compliancewith Zhirnov et al. (2009).

In conclusion, Caco-2 cells were found readily permissive toLPAI viruses. Their sensitivity to LPAI viruses was found compa-rable to embryonated chicken eggs, but more sensitive than MDCKcells without supplementary trypsin to MDCK cell culture medium.Caco-2 cells support multicycle replication of and plaque pro-duction by LPAI viruses without supplementary trypsin However,addition of trypsin at the rate of 2 �g/ml in cell culture mediumboost viral titer yield. The cleavage of HA0 occurred intracellularly.Virus isolation rate from fecal and swab samples were found to belower than that in embryonated chicken eggs. Also, Caco-2 cellswere found not to generate cell mediated viral variants, at leastuntil 10 passages. Finally, the Caco-2 cell line may be regarded asuseful for AIV study and cultivation.

Acknowledgement

This study was supported in part by a grant from Ministry ofAgriculture, Forestry, and Fisheries (MAFF), Japan.

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