Journal of Medical Virology 84:1304–1311 (2012) Molecular Characterization and Phylogenetic Analysis of Human Parainfluenza Virus Type 3 Isolated From Saudi Arabia Fahad N. Almajhdi, 1,2 * Mohamed S. Alshaman, 1 and Haitham M. Amer 1,3 1 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia 2 Center of Excellence in Biotechnology Research, College of Science, King Saud University, Riyadh, Saudi Arabia 3 Faculty of Veterinary Medicine, Department of Virology, Cairo University, Giza, Egypt Human parainfluenza virus 3 (HPIV-3) is a lead- ing cause of respiratory disease in children worldwide. Previous sequence analyses of the entire virus genome, among different HPIV-3 strains, demonstrated that HN is the most vari- able gene. There is a dearth of data on HPIV-3 strains circulating in Saudi Arabia. In this re- port, HPIV-3 was screened in nasopharyngeal aspirates collected from hospitalized children with acute respiratory disease during two suc- cessive seasons (2007/08 and 2008/09) using nested RT-PCR. Out of 73 samples collected during 2007/08, seven (9.59%) were positive; while 3 out of 107 samples collected during 2008/09 (2.8%) were positive. Virus isolation in cell culture was successful using HEp2, but not Vero cells. The identity of the isolated viruses was confirmed using immunofluorescence and neutralization assays. To elucidate the genetic characteristics and phylogeny of Saudi HPIV-3 strains, the complete HN gene sequence of two selected Saudi strains was analyzed in compari- son to 20 strains isolated by others from differ- ent countries worldwide. Both strains showed the highest degree of sequence homology with Indian strains, followed by Chinese and most Japanese strains. Phylogenetic analysis con- firmed that these strains fell into a distinct Asian lineage. This study is the first in Saudi Arabia to recover HPIV-3 isolates of confirmed identity, and to generate sequence data that may help in understanding virus diversity and evolution. J. Med. Virol. 84:1304–1311, 2012. ß 2012 Wiley Periodicals, Inc. KEY WORDS: Asian lineage; HPIV-3; hemag- glutinin–neuramindase; phylo- geny; sequence analysis; Saudi Arabia INTRODUCTION Human parainfluenza virus 3 (HPIV-3) is a major cause of respiratory tract illness in infants and young children worldwide [Henrickson, 2003]. All children experience at least one HPIV-3 infection by the age of 5, and reinfection may occur throughout the life as a result of incomplete immunity [Glezen et al., 1984]. The virus is associated with a wide variety of respira- tory tract affections, but most frequently with croup and pneumonia. Annual epidemics of HPIV-3 infec- tion are responsible for considerable economic losses as a result of hospitalization, medication costs, work and school absence, and mortalities. In United States alone, the figure of hospitalization due to HPIV-3 infections constitutes 11.5% of all pediatric respiratory tract hospitalizations [Karron and Collins, 2006]. HPIV-3 is an enveloped virus that belongs to the genus Respirovirus of the family Paramyxoviridae [Fauquet et al., 2005]. The virus genome is a single non-segmented negative strand of RNA that encodes six structural proteins (3 0 -N-P-M-F-HN-L-5 0 ). Viral RNA, together with nucleocapsid (N), phosphoprotein (P), and large (L) proteins, forms a helical nucleocap- sid, which is packaged into the viral envelope. Two glycosylated proteins hemagglutinin–neuramindase Grant sponsor: Center of Excellence in Biotechnology Research, King Saud University; Grant number: CEBR-GSRP 16/2012. The authors declare that they have no conflicts of interest. *Correspondence to: Fahad N. Almajhdi, Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box, 2455, Riyadh 11451, Kingdom of Saudi Arabia. E-mail: majhdi@ksu.edu.sa Accepted 23 April 2012 DOI 10.1002/jmv.23326 Published online in Wiley Online Library (wileyonlinelibrary.com). ß 2012 WILEY PERIODICALS, INC.