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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
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Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5
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Dorra Rezig ∗ , Henda Touzi, Zina Meddeb, Henda Triki Laboratory of Clinical Virology, WHO Regional Reference Laboratory on Poliomyelitis and Measles, Institut Pasteur de Tunis, Tunisia
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Article history: Received 27 November 2013 Received in revised form 7 April 2014 Accepted 6 June 2014 Available online xxx
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Keywords: Human cosavirus Picornaviridae Cell culture Cytopathic effect MRC5
Human cosaviruses (HCoSVs) are newly discovered viruses in Picornaviridae family. Up-to-date, most published studies reported HCoSV detection using molecular techniques and genetic characterization of the virus. Nevertheless, no laboratory has yet reported the replication of these viruses in cultured cell lines. In the present work, the propagation of HCoSV strains isolated from human fecal specimens in MRC5 cell line and their induced cytopathic effects (CPE) was studied. The first sign of virus growth was observed 24–48 h after inoculation. The cells rounded up and clumped together rapidly; empty areas became visible and, on the third day of CPE, a remarkable decrease in live cells was observed. This represents the first report on in vitro model of HCoSV replication which opens up opportunities for future investigations of these new viruses. © 2014 Published by Elsevier B.V.
The advent of metagenomics and the recent advances in sequencing technology have enabled the discovery of new human pathogens, particularly abundant in fecal specimens, such as the Human cosaviruses (HCoSVs). These new viruses are members of a new genus in the Picornaviridae family named Cosavirus (common stool-associated picornavirus) (Kapoor et al., 2008; Tapparel et al., 2013). According to the few published studies, HCoSVs are widespread geographically, detected with similar frequency in symptomatic and asymptomatic subjects, which makes it difficult to establish a disease association. This might be due to the existence of several species and serotypes within the same genus, or to a variable clinical presentation including a high proportion of asymptomatic forms, as observed with most Enteroviruses (Pallansch and Roos, 2001). Based on VP1 sequences, the Cosavirus genus is currently classified into five species (A–E). HCoSV-A and -D are the most prevalent with 24 and 5 different genotypes, respectively (Kapusinszky et al., 2012). The HCoSV genome is about 7.6 kb long and has the typical picornavirus genome organization, with the exception of the lack of a leader (L) sequence. The genome consists in four structural viral proteins (VP4, VP2, VP3, VP1), and nine non-structural
∗ Corresponding author at: Laboratory of Clinical Virology – Institut Pasteur, 13, Place Pasteur, BP 74, 1002 Tunis – Belvédère, Tunisia. Tel.: +216 71 783 022; fax: +216 71 791 833. E-mail address: dor [email protected] (D. Rezig).
proteins (2A1, 2A2, 2B, 2C, 3A, 3B1, 3B2, 3C, and 3D) (Kapoor et al., 2008). HCoSVs are usually detected by RT-PCR amplification in the 5 non-coding (5 NC) region of the genome. Sequencing of the VP1 and the 3D genomic regions is used to confirm the species and to determine the HCoSV genotype (Dai et al., 2010; Holtz et al., 2008; Kapoor et al., 2008; Khamrin et al., 2012; Stöcker et al., 2012). Upto-date, most published studies reported HCoSV detection using molecular techniques and genetic characterization of the detected viruses (Blinkova et al., 2009; Kapusinszky et al., 2012; Maan et al., 2013). To our knowledge, the propagation of these viruses in cultured cell lines has not yet been reported. The present study reports the replication in MRC5 cell line and the induced cytopathic effect (CPE) of HCoSV strains, isolated from human fecal specimens. The HCoSV strains obtained in this study were isolated from the stool samples of one patient with Acute Flaccid Paralysis or AFP (S231TUN2006) and two healthy individuals from the contacts of other AFP cases (E11TUN2012 and E64TUN2012). The three individuals were investigated as part of the poliovirus surveillance program in Tunisia; their stool samples showed no CPE on the WHO-recommended cell lines to detect polioviruses: RD, L20B and Hep2C. The presence of HCoSVs was first detected by PCR, using an “in-house” protocol amplifying in the 5 NC region of the viral genome and previously published primers (Kapoor et al., 2008). Briefly, after RNA extraction using the QIAmp viral RNA minikit (Qiagen, Hilden, Germany), cDNA synthesis was performed in a reaction mixture containing 5 l of RNA extract, 50 M of reverse primer
http://dx.doi.org/10.1016/j.jviromet.2014.06.011 0166-0934/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Rezig, D., et al., Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.06.011
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Fig. 1. Cytopathic effect caused by HCoSV. Non-infected human embryonic lung cells (A and B), human embryonic lung cells on 24 h post-infection (C), human embryonic lung cells on 48 h post-infection (D), human embryonic lung cells on 72 h post-infection at a magnification of 10× (E) and 20× (F). Visible empty areas are indicated by arrows. Photos of MRC5 cell cultures non-infected and infected were taken at a magnification of 10× (A–E) except (F) at magnification of 20×.
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Co-5NC-R2 (5 -GTACCTTCAGGACATCTTTGG-3 ), 40 U of RNAsin (Amersham Pharmacia Biotech, Piscataway, NJ, USA), 2 l of 5× concentrated reverse transcriptase buffer (250 mM Tris–HCl; pH 8.5, 150 mM KCl, 5 mM dithiothreitol, 40 mM MgCl2 ), 10 mM dNTPs (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and 5 U of Avian Murine Leukemia reverse transcriptase (Boerhinger, Ingelheim, Germany) in a total volume of 10 l. The mixture was incubated at 42 ◦ C for 45 min. The total volume of cDNA was then used for PCR amplification in the presence of 5 l of amplification buffer 10× concentrated (Amersham Pharmacia Biotech, USA), 50 M of Co-5NC-R2 reverse primer, 50 M of Co-5NC-F1 forward primer (5 -CGTGCTTTACACGGTTTTTGA-3 ), 2.5 U of Taq polymerase (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and 20 mM of each dNTP(Amersham Pharmacia Biotech, Piscataway, NJ, USA). The reaction mixture was then subjected to 40 cycles of 1 min denaturation at 94 ◦ C, 1 min annealing at 53 ◦ C, and 1 min extension at 72 ◦ C, with a final elongation step of 5 min at 72 ◦ C. One
microlitre of product from the first round PCR was then used as template in the second PCR round. The nested PCR was performed with 5 l of amplification buffer 10× concentrated (Amersham Pharmacia Biotech, Piscataway, NJ, USA), 50 M of the reverse primer Co-5NC-R3 (5 -GTCCTTTCGGACAGGGCTTT-3 ), 50 M of forward primer Co-5NC-F2 (5 -ACGGTTTTTGAACCCCACAC-3 ), 2.5 U of Taq polymerase (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and 20 mM of each dNTP (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The reaction mixture was then subjected to 40 cycles of 45 sec denaturation at 94 ◦ C, 45 s annealing at 54 ◦ C, and 45 s extension at 72 ◦ C, with a final elongation step of 5 min at 72 ◦ C. The amplification products of 316 pb were all run on a 2% agarose gel stained with ethidium bromide. For virus isolation on cell culture, human embryonic lung cell line (MRC5) (European Collection of Cell Cultures-lot 03/E/02703/06/03, P+15) was maintained in minimum essential medium (MEM) (GIBCO-Invitrogen, NY, USA) supplemented with 10%
Please cite this article in press as: Rezig, D., et al., Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.06.011
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Fig. 2. Ethidium bromide-stained 2% agarose gel showing the results of the RT nested PCR assay in the 5 NC region of the viral genome. Amplified product of 316 bp bands is shown for HCoSV strains. M: molecular weight marker (Roche Diagnostics, USA); N, negative control; 1–3, represent HCoSV isolates obtained after cell culture amplification; and 1 -3 , represent HCoSV from clinical specimens: Laboratory codes: 1 and 1 , E11TUN2012; 2 and 2 , E64TUN2012; 3 and 3 , S231TUN2006; a weak positive band (lane 1 ) was visible in the gel but it does not appear in the photo.
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(v/v) Fetal Bovine Serum (FBS) (GIBCO-Invitrogen, NY, USA) and 2% penicillin-streptomycin-gentamycine (Pharmadrug Production, Hamburg, Germany) at 37 ◦ C with 5% CO2 . Cell culture tubes with confluent MRC5 cell monolayer were prepared and inoculated with 200 l of stool suspension, prepared in PBS/chloroform according to the WHO-recommended protocol for polio surveillance (WHO, 2004). The cell culture media was first discarded and the stool suspension was then incubated, without media, 1 h at 37 ◦ C for adsorption. A cell culture tube with confluent MRC5 cell monolayer was used as a negative control; the stool suspension was replaced by 200 l of cell culture medium and the monolayer was incubated in the same conditions. After adsorption, 2 ml of maintenance medium were added to each tube and 200 l of cell supernatant were collected for HCoSV detection and quantification. The cells were then placed in the incubator at 37 ◦ C and 5% CO2 and checked for CPE daily using an inverted microscope (Laica, Japan). The first sign of virus growth was observed after 24 h for two samples (S231TUN2006 and E64TUN2012) and 48 h for the third sample (E11TUN2012) while no morphological changes were observed for the uninfected MRC5 cells in the negative control tube. The CPE was similar in the 3 inoculated tubes; Fig. 1 shows the one observed with sample S231TUN2006. Twenty-four hours after inoculation, the cells round up and clump together but are still covering the surface of the tube (Fig. 1C). In the second day of CPE, empty areas become visible (indicated by arrows, Fig. 1D) and rounded-off, refractive and small-sized cells are observed. On the third day of CPE, a higher number of floating cells in the media is detected with a remarkable decrease in live cells (Fig. 1E and F). To confirm the presence of HCoSVs, RNA was extracted from 200 l of cell supernatant and tested by RT-PCR using the same HCoSV “in-house” PCR test described above. Fig. 2 shows the amplification products obtained at 3-days of CPE (Lanes 1, 2 and 3) and those obtained from the cell supernatant collected immediately after inoculation (Lanes 1 , 2 and 3 ). The amplification bands were stronger after 3 days of culture. To further confirm virus amplification on MRC5 cell between the first day, immediately after inoculation, and the third day, when the CPE was visible, cell supernatant at day 1 and at day 3 were tested comparatively by an “in-house” real-time PCR protocol: RNA was first extracted from 200 l and cDNA was obtained by reverse transcription using 5 l RNA extract and the reverse primer Co-5NC-R2; according to the protocol described above. SYBR Green real-time amplifications were then performed using 10 M of reverse and forward primers (Co-5NC-R2 and Co-5NC-F1) and 8 l of cDNA in a final volume of 25 l. The reaction tubes were then placed in an ABI-7500
real-time PCR System, the thermal profile was 2 min at 50 ◦ C, 10 min at 95 ◦ C followed by 40 cycles of 15 s at 94 ◦ C and 1 min at 60 ◦ C. The results showed higher virus amounts in the cell supernatants collected at day3, in comparison to those collected at day1, for the three samples, with a Ct ranging from 3.34 to 4.61 (Table 1). This study shows that HCoSVs replicate in MRC5 but not in RD, Hep2C and L20B cells. However, other cell lines may be permissive to these viruses. The CPE was relatively rapid for the three tested HCoSV positive samples: 24 h for samples S231TUN2006 and E64TUN2012, 48 h for sample E11TUN2012. The CPE was similar for the 3 studied viral strains but different from the CPE reported for other picornavirus (Gabriel et al., 1964; Pal et al., 1963). Sample S231TUN2006 was previously sequenced and identified as belonging to HCoSV-A genetic species (Kapusinszky et al., 2012). Also, we have sequenced the 3D genomic region of sample E64TUN2012 (data not shown) and found it related to an E/D recombinant, previously-described by the same authors (Kapusinszky et al., 2012). This indicates that at least HCoSVs of genetic species A, D and/or E can replicate on cell culture. Further investigations are needed to check if the other HCoSV species are able to replicate on cell culture as well and to study their replication features. In conclusion, although most previous reports showed sensitive detection of HCoSVs using molecular techniques and accurate genetic characterization (Blinkova et al., 2009; Kapusinszky et al., 2012; Maan et al., 2013), no evidence was reported for in vitro propagation of these viruses. This study shows that HCoSV viruses are able to replicate in MRC5 cells which may represent a relevant in vitro model to better characterize these viruses. Molecular techniques may be more convenient to detect the viruses for diagnostic purposes, especially in terms of rapidity. However, viral propagation in cell culture can provide valuable information especially for newly discovered strains and contributes to a better knowledge of their infectivity, virulence and replication; it can also establish virus banks ready to be used when ever needed.
Table 1 Results in Ct of the real-time PCR performed on cell supernatants at day 1 and day 3 post infection.
Cell supernatant at day1 Cell supernatant at day3 Threshold Ct
S231TUN2006
E11TUN2012
E64TUN2012
34.63 30.99 0.012091 3.64
34.78 30.17 0.016296 4.61
33.24 29.90 0.010644 3.34
Please cite this article in press as: Rezig, D., et al., Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.06.011
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This study was supported by the Tunisian Ministry for High Education and Scientific Reasearch (LR11-IPT09). We are grateful to Khaled Trabelsi and Semi Majoul from the Laboratoire de recherche et développement – Institut Pasteur de Tunis for the supplies of MRC5 cells used in this investigation. We would like to thank Dr Naziha Marrakchi from the Laboratoire des Venins et Molecules Thérapeutiques- Institut Pasteur de Tunis for the photographic grips.
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References
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Blinkova, O., Rosario, K., Li, L., Kapoor, A., Slikas, B., Bernardin, F., Breitbart, M., Delwart, E., 2009. Frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the United States. J. Clin. Microbiol. 47 (11), 3507–3513. Dai, X.Q., Hua, X.G., Shan, T.L., Delwart, E., Zhao, W., 2010. Human cosavirus infections in children in China. J. Clin. Virol. 48 (3), 228–229. Gabriel, G.C., Rifkind, R.A., Howe, C., Rose, H.M., 1964. A description of ECHO-9 virus infection in cultured cells. Am. J. Pathol. 44 (1), 1–27. Holtz, L.R., Finkbeiner, S.R., Kirkwood, C.D., Wang, D., 2008. Identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea. Virol. J. 5, 159. Kapoor, A., Victoria, J., Simmonds, P., Slikas, E., Chieochansin, T., Naeem, A., Shaukat, S., Sharif, S., Alam, M.M., Angez, M., Wang, C., Shafer, R.W., Zaidi, S.,
Delwart, E., 2008. A highly prevalent and genetically diversified Picornaviridae genus in South Asian children. Proc. Natl. Acad. Sci. U. S. A. 105 (51), 20482– 20487. Kapusinszky, B., Phan, T.G., Kapoor, A., Delwart, E., 2012. Genetic diversity of the genus Cosavirus in the family Picornaviridae: a new species, recombination, and 26 new genotypes. PLoS One 7 (5), e36685. Khamrin, P., Chaimongkol, N., Malasao, R., Suantai, B., Saikhruang, W., Kongsricharoern, T., Ukarapol, N., Okitsu, S., Shimizu, H., Hayakawa, S., Ushijima, H., Maneekarn, N., 2012. Detection and molecular characterization of cosavirus in adults with diarrhea, Thailand. Virus Genes 44 (2), 244– 246. Maan, H.S., Chowdhary, R., Shakya, A.K., Dhole, T.N., 2013. Genetic diversity of cosaviruses in nonpolio acute flaccid paralysis cases of undefined etiology, Northern India, 2010–2011. J. Clin. Virol. 58 (1), 183–187. Pal, S.R., McQuillin, J., Gardner, P.S., 1963. A comparative study of susceptibility of primary monkey kidney cells, Hep2 cells and HeLa cells to a variety of faecal viruses. J. Hyg. 61, 493–498. Pallansch, M.A., Roos, R.P., 2001. Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology. Lippincott Williams and Wilkins, Philadelphia, pp. 723–775. Stöcker, A., Souza, B.F., Ribeiro, T.C., Netto, E.M., Araujo, L.O., Corrêa, J.I., Almeida, P.S., de Mattos, A.P., Ribeiro Hda Jr., C., Pedral-Sampaio, D.B., Drosten, C., Drexler, J.F., 2012. Cosavirus infection in persons with and without gastroenteritis, Brazil. Emerg. Infect. Dis. 18 (4), 656–659. Tapparel, C., Siegrist, F., Petty, T.J., Kaiser, L., 2013. Picornavirus and enterovirus diversity with associated human diseases. Infect. Genet. Evol. 14, 282– 293. WHO (World Health Organization), 2004. Manual for Virological Investigation of Poliomyelitis. WHO, Geneva.
Please cite this article in press as: Rezig, D., et al., Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.06.011
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ARTICLE IN PRESS
VIRMET 12552 1–4
Journal of Virological Methods xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
1
Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5
2
3
4 5
Q1
Dorra Rezig ∗ , Henda Touzi, Zina Meddeb, Henda Triki Laboratory of Clinical Virology, WHO Regional Reference Laboratory on Poliomyelitis and Measles, Institut Pasteur de Tunis, Tunisia
6
a b s t r a c t
20 7 8 9 10 11 12
Article history: Received 27 November 2013 Received in revised form 7 April 2014 Accepted 6 June 2014 Available online xxx
13 14 15 16 17 18 19
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Keywords: Human cosavirus Picornaviridae Cell culture Cytopathic effect MRC5
Human cosaviruses (HCoSVs) are newly discovered viruses in Picornaviridae family. Up-to-date, most published studies reported HCoSV detection using molecular techniques and genetic characterization of the virus. Nevertheless, no laboratory has yet reported the replication of these viruses in cultured cell lines. In the present work, the propagation of HCoSV strains isolated from human fecal specimens in MRC5 cell line and their induced cytopathic effects (CPE) was studied. The first sign of virus growth was observed 24–48 h after inoculation. The cells rounded up and clumped together rapidly; empty areas became visible and, on the third day of CPE, a remarkable decrease in live cells was observed. This represents the first report on in vitro model of HCoSV replication which opens up opportunities for future investigations of these new viruses. © 2014 Published by Elsevier B.V.
The advent of metagenomics and the recent advances in sequencing technology have enabled the discovery of new human pathogens, particularly abundant in fecal specimens, such as the Human cosaviruses (HCoSVs). These new viruses are members of a new genus in the Picornaviridae family named Cosavirus (common stool-associated picornavirus) (Kapoor et al., 2008; Tapparel et al., 2013). According to the few published studies, HCoSVs are widespread geographically, detected with similar frequency in symptomatic and asymptomatic subjects, which makes it difficult to establish a disease association. This might be due to the existence of several species and serotypes within the same genus, or to a variable clinical presentation including a high proportion of asymptomatic forms, as observed with most Enteroviruses (Pallansch and Roos, 2001). Based on VP1 sequences, the Cosavirus genus is currently classified into five species (A–E). HCoSV-A and -D are the most prevalent with 24 and 5 different genotypes, respectively (Kapusinszky et al., 2012). The HCoSV genome is about 7.6 kb long and has the typical picornavirus genome organization, with the exception of the lack of a leader (L) sequence. The genome consists in four structural viral proteins (VP4, VP2, VP3, VP1), and nine non-structural
∗ Corresponding author at: Laboratory of Clinical Virology – Institut Pasteur, 13, Place Pasteur, BP 74, 1002 Tunis – Belvédère, Tunisia. Tel.: +216 71 783 022; fax: +216 71 791 833. E-mail address: dor [email protected] (D. Rezig).
proteins (2A1, 2A2, 2B, 2C, 3A, 3B1, 3B2, 3C, and 3D) (Kapoor et al., 2008). HCoSVs are usually detected by RT-PCR amplification in the 5 non-coding (5 NC) region of the genome. Sequencing of the VP1 and the 3D genomic regions is used to confirm the species and to determine the HCoSV genotype (Dai et al., 2010; Holtz et al., 2008; Kapoor et al., 2008; Khamrin et al., 2012; Stöcker et al., 2012). Upto-date, most published studies reported HCoSV detection using molecular techniques and genetic characterization of the detected viruses (Blinkova et al., 2009; Kapusinszky et al., 2012; Maan et al., 2013). To our knowledge, the propagation of these viruses in cultured cell lines has not yet been reported. The present study reports the replication in MRC5 cell line and the induced cytopathic effect (CPE) of HCoSV strains, isolated from human fecal specimens. The HCoSV strains obtained in this study were isolated from the stool samples of one patient with Acute Flaccid Paralysis or AFP (S231TUN2006) and two healthy individuals from the contacts of other AFP cases (E11TUN2012 and E64TUN2012). The three individuals were investigated as part of the poliovirus surveillance program in Tunisia; their stool samples showed no CPE on the WHO-recommended cell lines to detect polioviruses: RD, L20B and Hep2C. The presence of HCoSVs was first detected by PCR, using an “in-house” protocol amplifying in the 5 NC region of the viral genome and previously published primers (Kapoor et al., 2008). Briefly, after RNA extraction using the QIAmp viral RNA minikit (Qiagen, Hilden, Germany), cDNA synthesis was performed in a reaction mixture containing 5 l of RNA extract, 50 M of reverse primer
http://dx.doi.org/10.1016/j.jviromet.2014.06.011 0166-0934/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Rezig, D., et al., Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.06.011
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Fig. 1. Cytopathic effect caused by HCoSV. Non-infected human embryonic lung cells (A and B), human embryonic lung cells on 24 h post-infection (C), human embryonic lung cells on 48 h post-infection (D), human embryonic lung cells on 72 h post-infection at a magnification of 10× (E) and 20× (F). Visible empty areas are indicated by arrows. Photos of MRC5 cell cultures non-infected and infected were taken at a magnification of 10× (A–E) except (F) at magnification of 20×.
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Co-5NC-R2 (5 -GTACCTTCAGGACATCTTTGG-3 ), 40 U of RNAsin (Amersham Pharmacia Biotech, Piscataway, NJ, USA), 2 l of 5× concentrated reverse transcriptase buffer (250 mM Tris–HCl; pH 8.5, 150 mM KCl, 5 mM dithiothreitol, 40 mM MgCl2 ), 10 mM dNTPs (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and 5 U of Avian Murine Leukemia reverse transcriptase (Boerhinger, Ingelheim, Germany) in a total volume of 10 l. The mixture was incubated at 42 ◦ C for 45 min. The total volume of cDNA was then used for PCR amplification in the presence of 5 l of amplification buffer 10× concentrated (Amersham Pharmacia Biotech, USA), 50 M of Co-5NC-R2 reverse primer, 50 M of Co-5NC-F1 forward primer (5 -CGTGCTTTACACGGTTTTTGA-3 ), 2.5 U of Taq polymerase (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and 20 mM of each dNTP(Amersham Pharmacia Biotech, Piscataway, NJ, USA). The reaction mixture was then subjected to 40 cycles of 1 min denaturation at 94 ◦ C, 1 min annealing at 53 ◦ C, and 1 min extension at 72 ◦ C, with a final elongation step of 5 min at 72 ◦ C. One
microlitre of product from the first round PCR was then used as template in the second PCR round. The nested PCR was performed with 5 l of amplification buffer 10× concentrated (Amersham Pharmacia Biotech, Piscataway, NJ, USA), 50 M of the reverse primer Co-5NC-R3 (5 -GTCCTTTCGGACAGGGCTTT-3 ), 50 M of forward primer Co-5NC-F2 (5 -ACGGTTTTTGAACCCCACAC-3 ), 2.5 U of Taq polymerase (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and 20 mM of each dNTP (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The reaction mixture was then subjected to 40 cycles of 45 sec denaturation at 94 ◦ C, 45 s annealing at 54 ◦ C, and 45 s extension at 72 ◦ C, with a final elongation step of 5 min at 72 ◦ C. The amplification products of 316 pb were all run on a 2% agarose gel stained with ethidium bromide. For virus isolation on cell culture, human embryonic lung cell line (MRC5) (European Collection of Cell Cultures-lot 03/E/02703/06/03, P+15) was maintained in minimum essential medium (MEM) (GIBCO-Invitrogen, NY, USA) supplemented with 10%
Please cite this article in press as: Rezig, D., et al., Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.06.011
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Fig. 2. Ethidium bromide-stained 2% agarose gel showing the results of the RT nested PCR assay in the 5 NC region of the viral genome. Amplified product of 316 bp bands is shown for HCoSV strains. M: molecular weight marker (Roche Diagnostics, USA); N, negative control; 1–3, represent HCoSV isolates obtained after cell culture amplification; and 1 -3 , represent HCoSV from clinical specimens: Laboratory codes: 1 and 1 , E11TUN2012; 2 and 2 , E64TUN2012; 3 and 3 , S231TUN2006; a weak positive band (lane 1 ) was visible in the gel but it does not appear in the photo.
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(v/v) Fetal Bovine Serum (FBS) (GIBCO-Invitrogen, NY, USA) and 2% penicillin-streptomycin-gentamycine (Pharmadrug Production, Hamburg, Germany) at 37 ◦ C with 5% CO2 . Cell culture tubes with confluent MRC5 cell monolayer were prepared and inoculated with 200 l of stool suspension, prepared in PBS/chloroform according to the WHO-recommended protocol for polio surveillance (WHO, 2004). The cell culture media was first discarded and the stool suspension was then incubated, without media, 1 h at 37 ◦ C for adsorption. A cell culture tube with confluent MRC5 cell monolayer was used as a negative control; the stool suspension was replaced by 200 l of cell culture medium and the monolayer was incubated in the same conditions. After adsorption, 2 ml of maintenance medium were added to each tube and 200 l of cell supernatant were collected for HCoSV detection and quantification. The cells were then placed in the incubator at 37 ◦ C and 5% CO2 and checked for CPE daily using an inverted microscope (Laica, Japan). The first sign of virus growth was observed after 24 h for two samples (S231TUN2006 and E64TUN2012) and 48 h for the third sample (E11TUN2012) while no morphological changes were observed for the uninfected MRC5 cells in the negative control tube. The CPE was similar in the 3 inoculated tubes; Fig. 1 shows the one observed with sample S231TUN2006. Twenty-four hours after inoculation, the cells round up and clump together but are still covering the surface of the tube (Fig. 1C). In the second day of CPE, empty areas become visible (indicated by arrows, Fig. 1D) and rounded-off, refractive and small-sized cells are observed. On the third day of CPE, a higher number of floating cells in the media is detected with a remarkable decrease in live cells (Fig. 1E and F). To confirm the presence of HCoSVs, RNA was extracted from 200 l of cell supernatant and tested by RT-PCR using the same HCoSV “in-house” PCR test described above. Fig. 2 shows the amplification products obtained at 3-days of CPE (Lanes 1, 2 and 3) and those obtained from the cell supernatant collected immediately after inoculation (Lanes 1 , 2 and 3 ). The amplification bands were stronger after 3 days of culture. To further confirm virus amplification on MRC5 cell between the first day, immediately after inoculation, and the third day, when the CPE was visible, cell supernatant at day 1 and at day 3 were tested comparatively by an “in-house” real-time PCR protocol: RNA was first extracted from 200 l and cDNA was obtained by reverse transcription using 5 l RNA extract and the reverse primer Co-5NC-R2; according to the protocol described above. SYBR Green real-time amplifications were then performed using 10 M of reverse and forward primers (Co-5NC-R2 and Co-5NC-F1) and 8 l of cDNA in a final volume of 25 l. The reaction tubes were then placed in an ABI-7500
real-time PCR System, the thermal profile was 2 min at 50 ◦ C, 10 min at 95 ◦ C followed by 40 cycles of 15 s at 94 ◦ C and 1 min at 60 ◦ C. The results showed higher virus amounts in the cell supernatants collected at day3, in comparison to those collected at day1, for the three samples, with a Ct ranging from 3.34 to 4.61 (Table 1). This study shows that HCoSVs replicate in MRC5 but not in RD, Hep2C and L20B cells. However, other cell lines may be permissive to these viruses. The CPE was relatively rapid for the three tested HCoSV positive samples: 24 h for samples S231TUN2006 and E64TUN2012, 48 h for sample E11TUN2012. The CPE was similar for the 3 studied viral strains but different from the CPE reported for other picornavirus (Gabriel et al., 1964; Pal et al., 1963). Sample S231TUN2006 was previously sequenced and identified as belonging to HCoSV-A genetic species (Kapusinszky et al., 2012). Also, we have sequenced the 3D genomic region of sample E64TUN2012 (data not shown) and found it related to an E/D recombinant, previously-described by the same authors (Kapusinszky et al., 2012). This indicates that at least HCoSVs of genetic species A, D and/or E can replicate on cell culture. Further investigations are needed to check if the other HCoSV species are able to replicate on cell culture as well and to study their replication features. In conclusion, although most previous reports showed sensitive detection of HCoSVs using molecular techniques and accurate genetic characterization (Blinkova et al., 2009; Kapusinszky et al., 2012; Maan et al., 2013), no evidence was reported for in vitro propagation of these viruses. This study shows that HCoSV viruses are able to replicate in MRC5 cells which may represent a relevant in vitro model to better characterize these viruses. Molecular techniques may be more convenient to detect the viruses for diagnostic purposes, especially in terms of rapidity. However, viral propagation in cell culture can provide valuable information especially for newly discovered strains and contributes to a better knowledge of their infectivity, virulence and replication; it can also establish virus banks ready to be used when ever needed.
Table 1 Results in Ct of the real-time PCR performed on cell supernatants at day 1 and day 3 post infection.
Cell supernatant at day1 Cell supernatant at day3 Threshold Ct
S231TUN2006
E11TUN2012
E64TUN2012
34.63 30.99 0.012091 3.64
34.78 30.17 0.016296 4.61
33.24 29.90 0.010644 3.34
Please cite this article in press as: Rezig, D., et al., Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.06.011
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G Model
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Acknowledgments
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This study was supported by the Tunisian Ministry for High Education and Scientific Reasearch (LR11-IPT09). We are grateful to Khaled Trabelsi and Semi Majoul from the Laboratoire de recherche et développement – Institut Pasteur de Tunis for the supplies of MRC5 cells used in this investigation. We would like to thank Dr Naziha Marrakchi from the Laboratoire des Venins et Molecules Thérapeutiques- Institut Pasteur de Tunis for the photographic grips.
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