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Journal of Clinical Microbiology, February 2009, p. 466-468, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.00892-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

In Vivo and In Vitro Alterations in Influenza A/H3N2 Virus M2 and Hemagglutinin Genes: Effect of Passage in MDCK-SIAT1 Cells and Conventional MDCK Cells{triangledown}

Danjuan Li,1* Reiko Saito,1 Yasushi Suzuki,1 Isamu Sato,2 Hassan Zaraket,1 Clyde Dapat,1 Isolde Ma Caperig-Dapat,1 and Hiroshi Suzuki1

Department of Public Health, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan,1 Yoiko Pediatric Clinic, Niigata, Japan2

Received 8 May 2008/ Returned for modification 18 July 2008/ Accepted 24 November 2008


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ABSTRACT
 
No mutations were detected in the hemagglutinin gene of influenza A/H3N2 virus isolates from patients undergoing short-term amantadine treatment. However, genetic changes occurred after serial passage in either MDCK or MDCK-SIAT1 cells. Our results showed that only a few mutations were observed in MDCK-SIAT1-passaged isolates in the presence of amantadine.


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TEXT
 
Viral resistance to amantadine emerged quickly in vivo when patients received amantadine for the treatment of influenza A virus infection (3, 5). The genetic basis of resistance to amantadine is associated with amino acid substitutions in the transmembrane region of the M2 gene (2). We documented a high frequency of amantadine-resistant influenza A/H3N2 virus isolates with a Ser-31-Asn mutation in the M2 gene and dual changes in the hemagglutinin (HA) gene at residues 193 and 225 (clade N lineage) (10). However, little is known about whether the HA changes (i) were synergistic with those occurring in the M2 gene in response to drug selection pressure or (ii) occurred separately and were randomly associated with fitness-improving mutations. To address this question, we analyzed changes in the M2 and HA genes of influenza A viruses from clinical samples and from the same isolates that were serially passaged in Madin-Darby canine kidney (MDCK) cells or SIAT1-transfected MDCK cells in the presence or absence of amantadine. The MDCK cell line is commonly used for influenza virus isolation, while the MDCK-SIAT1 cell line is a recently introduced MDCK variant characterized by an overexpression of sialyl-{alpha}2,6-galactose that improves the binding of human influenza A virus to the cell receptor (6).

Nasopharyngeal swabs were collected from patients with an influenza-like illness who visited a pediatric outpatient clinic in Niigata City, Japan, from 2000 to 2002. Samples were collected at the first visit and at the second visit after 3 to 5 days of amantadine treatment. One hundred microliters of each sample was inoculated into MDCK cells for virus isolation. Antigenic characterization was performed by hemagglutination inhibition test (12). Influenza A viruses were screened for amantadine susceptibility by the 50% tissue culture infective dose/0.2-ml method (5) and verified by M2 gene sequencing of the transmembrane region to a detect mutation at position 26, 27, 30, 31, or 34 that confers resistance (12). After screening, influenza A/H3N2 viruses that were originally amantadine sensitive and became amantadine resistant after drug treatment were selected and analyzed for this study (in vivo).

MDCK-SIAT1cells (kindly donated by Mikhail Matrosovich, Institute of Virology, Philipps University, Marburg, Germany) were passaged as described elsewhere (6). The selected parental amantadine-sensitive strains were inoculated into MDCK cells or MDCK-SIAT1 cells and sequentially passaged 10 times in the presence or absence of amantadine at a final concentration of 2.0 µg/ml. The viruses were analyzed after the 3rd and 10th passages in the absence or presence of amantadine in MDCK or MDCK-SIAT1 cells (in vitro). Viral RNA extraction and cDNA synthesis were performed as described elsewhere (1). After amplification of the M2 and HA genes, direct sequencing of PCR products was performed with an ABI 3100 DNA sequencer (11). The transmembrane region of the M2 channel protein and the coding regions of the HA1 domain (amino acid residues 1 to 329) and the HA2 domain (amino acid residues 1 to 208) were analyzed. No virus plaque purification and cloning of PCR products was performed prior to sequencing.

Our in vivo study showed that all of the virus isolates (n = 7) obtained from patients treated with amantadine possessed M2 gene mutations, whereas HA changes were not observed after amantadine treatment (Table 1).


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  		TABLE 1. Sequence analysis of H3N2 variants selected in vivo and in vitro

Our in vitro study showed that all of the viruses developed M2 gene mutations after 10 passages in both MDCK and MDCK-SIAT1 cells in the presence of amantadine, but the M2 mutation sites differed between the two cell lines for five of the isolates (Table 1). On the other hand, no mutations were observed in amantadine-free cultures after 10 passages, except for one isolate that showed an A30T substitution in M2 (conferring amantadine resistance) when grown in MDCK cells.

Analysis of the HA gene showed that six out of seven isolates developed mutations after three passages in MDCK cells, and eventually all of the isolates showed mutations in the HA1 and HA2 domains after 10 passages in the presence or absence of amantadine (Table 1). Most of the HA mutation sites and the type of amino acid substitutions were similar between the isolates passaged with or without amantadine and between 3rd- and 10th-passage isolates. However, the number of mutation sites increased after the 10th passage when cells were cultivated in the presence of amantadine.

HA changes were not observed in viruses after the 3rd passage in MDCK-SIAT1 cells and occurred in only one or two viruses after the 10th passage without or with amantadine, respectively (Table 1). The HA mutation sites and types of amino acid substitutions found were similar to those found in MDCK-passaged viruses but were far less numerous.

Our clinical surveillance results suggested that the short-term amantadine treatment did not drive HA mutations, and thus, the appearance of clade N was due to the combined events of reassortment (7, 13), amantadine-driven point mutations in the M2 gene, and fitness-improving mutations in the HA gene (11).

The HA gene was more variable in MDCK-passaged viruses than that in their MDCK-SIAT1-passaged counterparts. Mutations in amantadine-free culture accumulated mostly at the receptor-binding (RB) pocket in the HA1 subunit (residues 156, 220, and 229) (14, 15), whereas changes in the HA2 subunit did not have any functional significance. We assume that frequent mutations near RB sites can be attributed to adaptation of the viruses to MDCK cells, which express larger amounts of NeuAc{alpha}2,3Gal and smaller amounts of NeuAc{alpha}2,6Gal than do human airway epithelial cells (9). Egg-adapted influenza viruses also showed high specificities of binding to NeuAc{alpha}2,3Gal, presumably resulting from key amino acid changes at the RB site (at position 226) (4). On the other hand, MDCK-SIAT1 cells, engineered to overexpress NeuAc{alpha}2,6Gal, led to fewer HA mutations and could thus be more reliable for multipassage analysis of human influenza virus. Similar results for MDCK-SIAT1-passaged viruses were recently demonstrated by Oh et al., but different amino acid substitutions in the HA1 subunit were observed (8).

In our study, amantadine drove more HA1 mutations in both cell lines (more stable in MDCK-SIAT1 cells) than under amantadine-free conditions, and most of them were key amino acids at or near RB sites (positions 144, 156, 183, 186, 199, 220, 221, 226, 229, and 236) (13-15). Thus, amantadine may contribute to the appearance of a mutated HA gene with an RB site property altered by an unknown mechanism. While our clinical study demonstrated that amantadine did not affect the HA gene in the short term, the in vitro results suggested the possibility that longer exposure to the drug affects the HA gene in vivo, since MDCK-SIAT1 cell-passaged viruses also underwent changes in the HA gene in the presence of amantadine.

Given the small number of samples tested, our results elucidated the differences in the HA gene changes seen in vivo and in vitro in a comparison of two cell lines during the development of amantadine resistance. These results suggested that careful interpretation is needed after consecutive passages in MDCK cells, but MDCK-SIAT1 cells are more favorable for analysis of the RB domain and the molecular epidemiologic phylogeny of the HA gene.


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ACKNOWLEDGMENTS
 
We thank Mikhail Matrosovich (Institute of Virology, Philipps University, Marburg, Germany) for providing the SIAT1-transfected MDCK cells.

We declare that none of us have any conflict of interest.

This work was supported by Acute Respiratory Infections Panels, United States-Japan Cooperative Medical Science Program.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Public Health, Niigata University, Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Chuo-Ward, Niigata City, Niigata Prefecture 951-8510, Japan. Phone: 81-25-227-2129. Fax: 81-25-227-0765. E-mail: juanharbin2008{at}yahoo.co.jp Back

{triangledown} Published ahead of print on 17 December 2008. Back


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Journal of Clinical Microbiology, February 2009, p. 466-468, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.00892-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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