Protein-peptide arrays for detection of specific anti-hepatitis D virus (HDV) genotype 1, 6, and 8 antibodies among HDV-infected patients by surface plasmon resonance imaging.

Liver diseases linked to hepatitis B-hepatitis D virus co- or superinfections are more severe than those during hepatitis B virus (HBV) monoinfection. The diagnosis of hepatitis D virus (HDV) infection therefore remains crucial in monitoring patients but is often overlooked. To integrate HDV markers into high-throughput viral hepatitis diagnostics, we studied the binding of anti-HDV antibodies (Abs) using surface plasmon resonance imaging (SPRi). We focused on the ubiquitous HDV genotype 1 (HDV1) and the more uncommon African-HDV6 and HDV8 genotypes to define an array with recombinant proteins or peptides. Full-length and truncated small hepatitis D antigen (S-HDAg) recombinant proteins of HDV genotype 1 (HDV1) and 11 HDV peptides of HDV1, 6, and 8, representing various portions of the delta antigen were grafted onto biochips, allowing SPRi measurements to be made. Sixteen to 17 serum samples from patients infected with different HDV genotypes were injected onto protein and peptide chips. In all, Abs against HDV proteins and/or peptides were detected in 16 out of 17 infected patients (94.12%), although the amplitude of the SPR signal varied. The amino-terminal part of the protein was poorly immunogenic, while epitope 65-80, exposed on the viral ribonucleoprotein, may be immunodominant, as 9 patient samples led to a specific SPR signal on peptide 65 type 1 (65#1), independently of the infecting genotype. In this pilot study, we confirmed that HDV infection screening based on the reactivity of patient Abs against carefully chosen HDV peptides and/or proteins can be included in a syndrome-based viral hepatitis diagnostic assay. The preliminary results indicated that SPRi studying direct physical HDAg-anti-HDV Ab interactions was more convenient using linear peptide epitopes than full-length S-HDAg proteins, due to the regeneration process, and may represent an innovative approach for a hepatitis syndrome-viral etiology-exploring array.

Blocking human enterovirus 71 replication by targeting viral 2A protease.

OBJECTIVES: Human enterovirus 71 (EV-71), a member of the Enterovirus genus, constitutes a major public health issue in the Asia-Pacific region, where it is associated with several severe neurological complications. There is currently no effective vaccine or antiviral against EV-71. The aim of this study was to determine whether the six amino acid peptide LVLQTM, which was previously shown to inhibit human rhinovirus (HRV) 2A protease (2A(pro)) activity in vitro and HRV replication in vivo in mice, could be of more general use against enteroviruses and more particularly against EV-71. METHODS: To investigate whether the LVLQTM peptide was a pseudosubstrate of EV-71 2A(pro), a recombinant luciferase containing the LVLQTM sequence was designed so that recognition of this sequence by 2A(pro) led to luciferase activation. Direct interaction between EV-71 2A(pro) and the LVLQTM peptide was further confirmed by isothermal titration calorimetry. We then tested the effects of the peptide on EV-71 2A(pro) cleavage activity and EV-71 replication in HeLa cells. RESULTS: We showed that the LVLQTM peptide behaved as an effective substrate analogue of EV-71 2A(pro), which binds into the active site of the protease with a dissociation rate constant of 9.6 µM. Moreover, LVLQTM significantly inhibited eIF4G cleavage activity of 2A(pro) as well as EV-71 replication in HeLa cells. CONCLUSIONS: This study demonstrates that the LVLQTM peptide that has previously been shown to inhibit HRV replication is also an effective inhibitor of EV-71 2A(pro) and therefore of EV-71 replication, opening new doors in the development of new antivirals against EV-71.

Effects of mutations on herpes simplex virus 1 thymidine kinase functionality: an in vitro assay based on detection of monophosphate forms of acyclovir and thymidine using HPLC/DAD.

Discrimination between the mutations responsible for drug resistance and those of UL23 TK gene polymorphism can be difficult. A non-isotopic method has been developed to assess TK functionality by measuring monophosphate forms of both acyclovir (ACV) and thymidine using HPLC/DAD. Phenotypes of TKs could thus be characterized as TK altered (P84L, A189V, L227F), TK deficient (G200S, L291P) or TK partial (R163H). A reliable link between HSV UL23 TK mutations and ACV resistance is necessary for developing a powerful genotyping tool to detect ACV resistance quickly in clinical samples.

Ex vivo and in vivo inhibition of human rhinovirus replication by a new pseudosubstrate of viral 2A protease.

Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2A(pro)) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model.