Recombinant Hepatitis A Virus Protease 3C (a.a.1643-1743)

Background: Hepatitis A virus (HAV) is a common cause of mild to acute hepatitis among children and adolescents, worldwide. Objectives: The aim of this study was to investigate the complete sequence of isolated HAV from a child with acute hepatitis. Methods: A serum sample was collected from a child with clinical sign and symptoms of acute hepatitis. Following RNA extraction, the genotype of virus was determined based on sequencing of VP1-2A region of the isolated HAV genome. The whole HAV genome was sequenced bythe nested polymerase chain reaction (PCR) using specific primers. The size of the complete length of isolated HAV genome, 5UTR (Untranslated Region), and 3UTR were determined. The amino acid sequencing of single polyprotein, including VP1 and VP3 regions, were analyzed using the BioEdit software. To evaluate the isolated HAV genome, phylogenetic tree was conducted to analyze the complete HAV Genome, 5UTR, and VP1-2A regions of the isolated HAV. Simplot and RDP4 analyses were accomplished to confirm recombination in the genome of the sequestered HAV strain in Ahvaz city. Results: The sequestered HAV/Ahvaz/Iran/2015 was recorded with an accession number Banklt 2063303 MG 546669 in GenBank. The complete HAV sequence of the isolated HAV comprised of full length of 7239 nt, 5'UTR 619 nt, 3'UTR 21nt, and an open reading frame (ORF) encoding single polypeptide of 2200 amino acids (6600 nt). The whole sequences of isolated HAVstrainAhvaz/Iran/2 015 showed 96% and 95% nucleotide identitywith prototype strain HAV1B isolated from Egypt (96%), SouthAfrica (95%), and HM175 (95%) strains, respectively. The complete amino acid sequence of the Vp1-2A region of the isolated HAV showed 100% identity with HM175 and 99% identity with isolated HAV from Egypt and South Africa. The detected HAV was identified as HAV genotype 1B. A mutation was observed in the amino acid sequences of VP3 region of the isolated HAV; this mutation showed substitution of isoleucine (I) with Arginine at position 433 amino acid sequence compared with the consensus sequences in amino acid of HM 175 strain. The results of RDP4 showed no evidence of recombination in the isolated strain HAV/Ahvaz/Iran/2 015 genome. Conclusions: The isolated HAV was genotypes B, comprised of full length of 7239 nt, 220 0 amino acid. The complete amino acid sequence VP1 region of the isolated HAV showed 100% homology identity with HM175 and 99% with isolated HAV in Egypt, South Africa. A mutation was observed in the amino acid sequences of VP3 region of the isolated HAV, this mutation showed substitution of isoleucine (I) with Arginine at position 433 amino acid sequence compared with the consensus sequences amino acid of HM-175 strain. No evidence of recombination was observed in the isolated strain HAV/Ahvaz/Iran/2015 genome.

The transmembrane glycoprotein N-cadherin (NCad) mediates cell-cell interactions found during mesenchymal condensation and chondrogenesis. Here, NCad-derived peptides (i.e., HAV) are incorporated into hyaluronic acid (HA) hydrogels with encapsulated mesenchymal stem cells (MSCs). Since the dose and timing of NCad signaling are dynamic, HAV peptide presentation is tuned via alterations in peptide concentration and incorporation of an ADAM10-cleavable domain between the hydrogel and the HAV motif, respectively. HA hydrogels functionalized with HAV result in dose-dependent increases in early chondrogenesis of encapsulated MSCs and resultant cartilage matrix production. For example, type II collagen and glycosaminoglycan production increase approximate to 9- and 2-fold with the highest dose of HAV (i.e., 2 x 10(-3)m), respectively, when compared to unmodified hydrogels, while incorporation of an efficient ADAM10-cleavable domain between the HAV peptide and hydrogel abolishes increases in chondrogenesis and matrix production. Treatment with a small-molecule ADAM10 inhibitor restores the functional effect of the HAV peptide, indicating that timing and duration of HAV peptide presentation is crucial for robust chondrogenesis. This study demonstrates a nuanced approach to the biofunctionalization of hydrogels to better emulate the complex cell microenvironment during embryogenesis toward stem-cell-based cartilage production.