Journal of Medical Virology 80:306–315 (2008) Genetic Analysis of the Hypervariable Region of the Human Astrovirus nsP1a Coding Region: Design of a New RFLP Typing Method Susana Guix, Santiago Caballero, Cristina Fuentes, Albert Bosch,* and Rosa M. Pinto ´ Enteric Virus Laboratory, Department of Microbiology, University of Barcelona, Barcelona, Spain Human astroviruses (HAstV) are causative agents of viral gastroenteritis worldwide. A hypervariable region (HVR) is located close to the C-terminus of the nsP1a, and recent data support the involvement of the HVR-containing nonstructural protein in viral RNA replication processes, suggesting a correlation between variability in this region and pathogenic properties. The HVR of the C-terminal nsP1a coding region of 104 wild-type and reference isolates of HAstV was sequenced. A phylogenetic analysis was performed to identify different genotypes, and a restriction fragment length poly- morphism (RFLP) method was designed. An extensive nucleotide and deduced amino acid sequence variability was observed, as well as many insertions and deletions that retained the reading frame. The resultant phylogenetic tree supported the subdivision of HAstV into the two previously described major genetic groups, genogroup A and B, and the identification of 12 genotypes (9 within genogroup A, and 3 within genogroup B), which could be identified by RFLP. A correlation analysis was performed between genotype information and viral load using information from 35 clinical samples. Significant differences were observed between the viral load in clinical samples and certain HAstV genotypes that belonged to the same serotype, confirming the influence of C-terminal nsP1a variability on the viral replication phenotype. The use of the new RFLP typing method based on the HVR of the C-terminal nsP1a coding region by diagnosticians would help to understand the relationship between different genotypes and the severity of the gastroenteritis. J. Med. Virol. 80:306–315, 2008. ß 2007 Wiley-Liss, Inc. KEY WORDS: astrovirus; hypervariable region; RFLP; genotyping INTRODUCTION Human astroviruses (HAstV) are causative agents of viral gastroenteritis worldwide mainly in children [Glass et al., 1996; Walter and Mitchell, 2003]. These nonenveloped positive-strand RNA viruses belong to the family Astroviridae, which includes both mammalian and avian astroviruses. The astrovirus genome consists of a 6.8-kb polyadenylated genome with three over- lapping open reading frames (ORFs); ORF1a and ORF1b are linked by a translational ribosomal frame- shifting and encode the viral protease and polymerase, respectively, and ORF2 encodes the structural proteins [Matsui and Greenberg, 2001]. On the basis of antigenic criteria, HAstV are divided into eight serotypes (HAstV-1 to HAstV-8). Phylogenetic analysis of different parts of the genome result in different genetic clustering [Belliot et al., 1997; Me ´ndez-Toss et al., 2000; Taylor et al., 2001; Lukashov and Goudsmit, 2002; Silva et al., 2006]. Most studies based on the capsid region suggest a high correlation between serotypes and genotypes, a relationship which allows determining serotype information by sequence analysis. The comparison of sequences from the capsid region gives rise to radial tree topologies with equidistant clustering of serotypes. Interestingly however, phylogenetic analysis of the well-conserved partial sequence close to the protease motif coding region, results in only two clearly differentiated genogroups, called genogroup A (HAstV-1 to HAstV-5, and HAstV-8) and genogroup B (HAstV-6 and HAstV-7). This different phylogenetic clustering may be the result of recombination events between structural and nonstructural coding regions [Belliot et al., 1997]. Grant sponsor: European Union (partial support); Grant number: SP22-CT-2004-502571; Grant sponsor: Generalitat de Catalunya; Grant number: 2005SGR00966; Grant sponsor: Centre de Refere `ncia de Biotecnologia de Catalunya (CeRBa); Grant sponsor: Generalitat de Catalunya. Santiago Caballero’s present address is Department of Research and Development, Instituto Grifols, S.A., Parets del Valle ´s, Spain. *Correspondence to: Albert Bosch, Department of Microbiology, University of Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain. E-mail: abosch@ub.edu Accepted 20 September 2007 DOI 10.1002/jmv.21058 Published online in Wiley InterScience (www.interscience.wiley.com) ß 2007 WILEY-LISS, INC.