Original Article
Copyright ©2014 Baishideng Publishing Group Inc. All rights reserved.
World J Virology. Nov 12, 2014; 3(4): 30-36
Published online Nov 12, 2014. doi: 10.5501/wjv.v3.i4.30
Substrate specificity of avian influenza H5N1 neuraminidase
Naruthai Onsirisakul, Shin-ichi Nakakita, Chompunuch Boonarkart, Alita Kongchanagul, Ornpreya Suptawiwat, Pilaipan Puthavathana, Krisada Chaichuen, Kanokwan Kittiniyom, Yasuo Suzuki, Prasert Auewarakul
Naruthai Onsirisakul, Kanokwan Kittiniyom, Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 73170, Thailand
Naruthai Onsirisakul, Chompunuch Boonarkart, Ornpreya Suptawiwat, Pilaipan Puthavathana, Prasert Auewarakul, Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol Univeristy, Bangkok 10700, Thailand
Shin-ichi Nakakita, Division of Functional Glycomics, Life Sciences Research Center, Kagawa University, Takamatsu 761-0793, Japan
Alita Kongchanagul, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom 73170, Thailand
Krisada Chaichuen, Faculty of Veterinary Science, Mahidol University, Nakorn Pathom 73170, Thailand
Yasuo Suzuki, College of Life and Health Sciences, Chubu University, Kasugai 487-8501, Japan
Author contributions: Onsirisakul N preformed the majority of experiments; Nakakita S performed HPLC; Boonarkart C, Suptawiwat O and Kongchanagul A helped to perform and proved the constructs; Puthavathana P, Chaichuen K provided the viruses; Suzuki Y provided the glycopolymer; Kittiniyom K provided the supervision; Auewarakul P supervised and edited manuscript.
Supported by Thailand Research Fund, the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative
Correspondence to: Prasert Auewarakul, Professor, Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol Univeristy, 2 Prannok Road, Bangkoknoi, Bangkok 10700, Thailand. sipaw@mahidol.ac.th
Telephone: +81-662-4198291 Fax: +81-662-4184148
Received: June 19, 2014
Revised: September 3, 2014
Accepted: October 14, 2014
Published online: November 12, 2014
Processing time: 147 Days and 21.7 Hours
Abstract

AIM: To characterise neuraminidase (NA) substrate specificity of avian influenza H5N1 strains from humans and birds comparing to seasonal influenza virus.

METHODS: Avian influenza H5N1 strains from humans and birds were recruited for characterising their NA substrate specificity by using a modified commercial fluorescence Amplex Red assay. This method can identify the preference of α2,6-linked sialic acid or α2,3-linked sialic acid. Moreover, to avoid the bias of input virus, reverse genetic virus using NA gene from human isolated H5N1 were generated and used to compare with the seasonal influenza virus. Lastly, the substrate specificity profile was further confirmed by high-performance liquid chromatography (HPLC) analysis of the enzymatic product.

RESULTS: The H5N1 NA showed higher activity on α2,3-linked sialic acid than α2,6-linked (P < 0.0001). To compare the NA activity between the H5N1 and seasonal influenza viruses, reverse genetic viruses carrying the NA of H5N1 viruses and NA from a seasonal H3N2 virus was generated. In these reverse genetic viruses, the NA activity of the H5N1 showed markedly higher activity against α2,3-linked sialic acid than that of the H3N2 virus, whereas the activities on α2,6-linkage were comparable. Interestingly, NA from an H5N1 human isolate that was previously shown to have heamagglutinin (HA) with dual specificity showed reduced activity on α2,3-linkage. To confirm the substrate specificity profile, HPLC analytic of enzymatic product was performed. Similar to Amplex red assay, H5N1 virus showed abundant preference on α2,3-linked sialic acid.

CONCLUSION: H5N1 virus maintains the avian specific NA and NA changes may be needed to accompany changes in HA receptor preference for the viral adaptation to humans.

Keywords: H5N1 avian influenza virus; Neuraminidase; Sialic acid; Adaptation; Substrate preference

Core tip: We analyzed neuraminidase (NA) substrate specificity of avian influenza H5N1 strains from humans and birds using a modified fluorescence assay, and the substrate specificity profile was further confirmed by high-performance liquid chromatography analysis of the enzymatic product. The H5N1 NA showed higher activity on α2,3-linkage. Interestingly, NA from an H5N1 human isolate that was previously shown to have heamagglutinin (HA) with dual specificity showed reduced activity on α2,3-linkage. These suggest that the H5N1 virus maintains the avian specific NA activity and that changes in the NA may be needed to compensate for changes in the HA specificity for the viral adaptation to human hosts.