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Journal of Virology, February 2009, p. 1572-1578, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01879-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

PB2 Protein of a Highly Pathogenic Avian Influenza Virus Strain A/chicken/Yamaguchi/7/2004 (H5N1) Determines Its Replication Potential in Pigs {triangledown}

Rashid Manzoor,1 Yoshihiro Sakoda,1 Naoki Nomura,1 Yoshimi Tsuda,1 Hiroichi Ozaki,2 Masatoshi Okamatsu,1 and Hiroshi Kida1,3*

Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan,1 Department of Veterinary Microbiology, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan,2 Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan3

Received 7 September 2008/ Accepted 24 November 2008

It has been shown that not all but most of the avian influenza viruses replicate in the upper respiratory tract of pigs (H. Kida et al., J. Gen. Virol. 75:2183-2188, 1994). It was shown that A/chicken/Yamaguchi/7/2004 (H5N1) [Ck/Yamaguchi/04 (H5N1)] did not replicate in pigs (N. Isoda et al., Arch. Virol. 151:1267-1279, 2006). In the present study, the genetic basis for this host range restriction was determined using reassortant viruses generated between Ck/Yamaguchi/04 (H5N1) and A/swine/Hokkaido/2/1981 (H1N1) [Sw/Hokkaido/81 (H1N1)]. Two in vivo-generated single-gene reassortant virus clones of the H5N1 subtype (virus clones 1 and 2), whose PB2 gene was of Sw/Hokkaido/81 (H1N1) origin and whose remaining seven genes were of Ck/Yamaguchi/04 (H5N1) origin, were recovered from the experimentally infected pigs. The replicative potential of virus clones 1 and 2 was further confirmed by using reassortant virus (rg-Ck-Sw/PB2) generated by reverse genetics. Interestingly, the PB2 gene of Ck/Yamaguchi/04 (H5N1) did not restrict the replication of Sw/Hokkaido/81 (H1N1), as determined by using reassortant virus rg-Sw-Ck/PB2. The rg-Sw-Ck/PB2 virus replicated to moderate levels and for a shorter duration than parental Sw/Hokkaido/81 (H1N1). Sequencing of two isolates recovered from the pigs inoculated with rg-Sw-Ck/PB2 revealed either the D256G or the E627K amino acid substitution in the PB2 proteins of the isolates. The D256G and E627K mutations enhanced viral polymerase activity in the mammalian cells, correlating with replication of virus in pigs. These results indicate that the PB2 protein restricts the growth of Ck/Yamaguchi/04 (H5N1) in pigs.

* Corresponding author. Mailing address: Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, hokkaido university, Kita 18 Nishi 9, Kita-Ku, Sapporo 060-0818, Japan. Phone: (81) 11-706-5207. Fax: (81) 11-706-5273. E-mail: kida{at}vetmed.hokudai.ac.jp

{triangledown} Published ahead of print on 3 December 2008.

Journal of Virology, February 2009, p. 1572-1578, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01879-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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