Journal of Medical Virology Hemagglutinin and Neuraminidase Genes of Influenza B Viruses Circulating in Riyadh, Saudi Arabia During 2010–2011: Evolution and Sequence Analysis Ghazanfar Ali, 1 Haitham M. Amer, 2,3 and Fahad N. Almajhdi 1,2 * 1 Center of Excellence in Biotechnology Research, King Saud University, Riyadh, Saudi Arabia 2 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia 3 Faculty of Veterinary Medicine, Virology Department, Cairo University, Giza, Egypt Influenza viruses are known as continuing threats to human public health every year worldwide. Evolutionary dynamics of influenza B viruses in humans are in a unique progres- sion having two lineages; B/Yam and B/Vic-like viruses, which are circulating simultaneously worldwide. There is a considerable lack of data on influenza B viruses circulating in Saudi Arabia. During the winter-spring season of 2010–2011, 80 nasopharyngeal aspirates were collected from hospitalized patients with flu-- like symptoms in Riyadh. Screening of samples by one-step RT-PCR identified three (3.8%) influenza B viruses. Sequencing of hemaggluti- nin (HA) and neuraminidase (NA) genes was performed to analyze influenza B viruses circu- lating in Riyadh as compared to the globally circulating strains. Several common and six unique amino acid substitutions were observed for both HA and NA genes of influenza B Saudi strains. Three unique substitutions (T182A, D196N, and K254R) were identified in HA gene of the B/Yam-like Riyadh strains. In NA gene, a unique common substitution (D53G) was found in all Riyadh strains, while two unique substitutions (L38P, G233R) were recognized only in B/Vic-like Riyadh strains. Riyadh strains were also found to contain N-glycosylation site in HA gene of both B/Vic and B/Yam lineages at positions 197–199 (NET) and 196–198 (NNK/ DNK), respectively. The significance of these mutations on the antigenicity of both lineages is discussed herein. The unique changes ob- served in HA and NA genes of influenza B Riyadh strains support strongly the need for continuous surveillance and monitoring of new evolving strains that might pose threat to the Saudi community. J. Med. Virol. # 2013 Wiley Periodicals, Inc. KEY WORDS: influenza B virus; DNA sequen- cing; hemagglutinin; neurami- nidase; phylogenetic Analysis; Saudi Arabia INTRODUCTION Influenza B viruses are members of family Ortho- myxoviridae, which contains a segmented negati- ve-sense RNA genome. The virus genome encodes for 11 proteins; namely polymerase PB1 (Seg-1), polymerase PB2 (Seg-2), polymerase PA (Seg-3), hemagglutinin (Seg-4), nucleoprotein (Seg-5), neur- aminidase (NA), and NB protein (Seg-6), matrix protein 1 and BM2 protein (Seg-7), non-structural protein 1 and non-structural protein 2 (Seg-8) [Lamb and Choppin, 1983]. Influenza viruses including type A and B are the main respiratory viruses that cause worldwide annual epidemics with high economic loss and approximately 250,000–500,000 deaths every year [WHO, 2003; Thompson et al., 2004]. Three phylogenet- ically and antigenically distinct influenza virus types; A, B, and C are circulating globally in the human Grant sponsor: Deanship of Scientific Research at King Saud University; Grant number: RGP-VPP-136. The present address of Ghazanfar Ali is Department of Biotechnology, University of Azad Jammu and Kashmir, 13100, Muzaffarabad, Pakistan  Correspondence to: Fahad N Almajhdi, Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. E-mail: vrg_ksu@yahoo.com Accepted 20 September 2013 DOI 10.1002/jmv.23819 Published online in Wiley Online Library (wileyonlinelibrary.com).  C 2013 WILEY PERIODICALS, INC.