Genetic analysis of the hypervariable region of the human astrovirus nsp1a coding region: Design of a new RFLP typing method

Abstract

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 polymorphism (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.

Human Astroviruses

“…Phylogenetic analysis of several partial sequences of ORF1a resulted in only two clearly separated groups, which have been called genogroup A (which comprises serotypes HAstV-1 to HAstV-5 and HAstV-8) and genogroup B (serotypes HAstV-6 and HAstV-7) (86, 175). As suggested, this different phylogenetic clustering may have resulted from recombination events between structural and nonstructural coding regions (86,175).…”

“…Sequence analysis of the hypervariable region in the nsP1a/4-coding region in ORF1a enables the additional differentiation of 12 genotypes, nine within genogroup A and three within genogroup B (86). In order to clearly distinguish them from ORF2-derived serotypes, the ORF1a-derived genotypes are named using roman numerals.…”

Evidence of a Recombinant Wild-Type Human Astrovirus Strain from a Kenyan Child with Gastroenteritis

“…As HAstVs are important pediatric diarrheal pathogens (7,17,20,29), the application of reliable diagnostic assays and characterization methods are essential for burden of disease investigations. An alternate typing scheme was proposed to address viral virulence studies (8), and a new classification scheme was proposed (12) to accommodate highly divergent HAstVs (AstV-MLB1, AstV-MLB2, VA1-3, HMO-A, and HMO-B) (4,5,12). The recent identification of novel HAstVs (4) and human, mink, and ovine-like AstVs (12) the presence of recombination and newly identified HAstVs, phylogenetic data may be skewed and misinterpreted.…”

The C-Terminal nsP1a Protein of Human Astrovirus Is a Phosphoprotein That Interacts with the Viral Polymerase

“…Additionally, an HVR, whose variability affects genome replication (8,9), is also present in this protein (22,25,29). Although the exact boundaries of the proteolytical cleavages of the ORF1a are not well identified, our initial working hypothesis was that a first cleavage at position Gln-567/Thr-568 (14) would render the nsP1a/4 protein of around 40 kDa and that further unknown processes of this protein would render smaller proteins.…”

“…In the nsP1a C-terminal end, named here the nsP1a/4 protein, several domains have been described: two coiled-coil regions, one coinciding with a death domain (DD), a nuclear localization signal (NLS), a putative viral genome-linked protein (VPg), and a hypervariable region (HVR) (1,6,11,13,22,25,29). Based on the HVR nucleotide genetic variability, 15 different nsP1a/4 protein HVR-derived genotypes (named using Roman numerals) have been established, and a restriction fragment length polymorphism (RFLP) typing method has been developed to consistently distinguish between genotypes (9). In addition, computational analyses of the nsP1a/4 coding region performed in our laboratory revealed the presence of acidic regions and of glutamine-and proline-rich regions, a death domain, and several putative O-glycosylation and phosphorylation sites (6)(7)(8).…”