Isolation and characterization of phage display-derived scFv antibodies against human parechovirus 1 VP0 protein.

Human parechoviruses (PeVs) are common viruses that are associated with a variety of diseases from mild gastrointestinal and respiratory symptoms to severe central nervous system infections. Until now there has not been antibodies for visualizing parechovirus infection. We used E. coli recombinant PeV-A1-VP0 protein as a target in phage display single chain variable fragment (scFv) antibody library panning. Three rounds of panning allowed identification and isolation of several candidate scFv clones, which tested positive in enzyme-linked immunosorbent assay (ELISA) against VP0. Three scFv clones (scFv-55, -59 and -71) with different CDR-3 sequences were further purified and tested in ELISA, Western blot and immunofluorescence microscopy (IFA) against a set of PeV-A1 isolates and a few isolates representing PeV types 2-6. In IFA, all three scFv binders recognized twenty PeV-A1 isolates. ScFv-55 and -71 also recognized clinical representatives of PeV types 1-6 both in IFA and in capture ELISA, while scFv-59 only recognized PeV-A1, -A2 and -A6. PeV-A1-VP0 (Harris strain) sequence was used to generate a peptide library, which allowed identification of a putative unique conformational antibody epitope with fully conserved flanking regions and a more variable core VVTYDSKL, shared between the scFv antibodies. Sequencing of the VP0 region of virus samples and sequence comparisons against parechoviral sequences in GenBank revealed 107 PeV-A1, -A3, -A8, -A17, -A (untyped) sequences with this exact epitope core sequence, which was most dominant among PeV-A1 isolates. These data suggest the first-time isolation of broad range phage display antibodies against human parechoviruses that may be used in diagnostic antibody development.

Macrodomain Binding Compound MRS 2578 Inhibits Alphavirus Replication.

Alphaviruses are positive-strand RNA viruses causing febrile disease. Macrodomain-containing proteins, involved in ADP-ribose-mediated signaling, are encoded by both host cells and several virus groups, including alphaviruses. In this study, compound MRS 2578 that targets the human ADP-ribose glycohydrolase MacroD1 inhibited Semliki Forest virus production as well as viral RNA replication and replicase protein expression. The inhibitor was similarly active in alphavirus trans-replication systems, indicating that it targets the viral RNA replication stage.

Monoclonal antibody against VP0 recognizes a broad range of human parechoviruses.

Parechoviruses (PeVs) are common viruses that cause mild gastrointestinal or respiratory symptoms to severe central nervous system infections. In infants, parechovirus infection is one of the leading causes of life-threatening viral disease. High-quality antibodies with broad binding specificities are essential to improve accurate parechovirus diagnosis in diagnostic laboratories. Such antibodies have potential in the development of rapid antigen detection assay against PeVs. In the present study, VP4 and VP2 genes from human parechovirus A1 (PeV-A1) were cloned and VP0 fusion protein produced to develop monoclonal antibodies against PeVs. Two pan-parechovirus antibodies, one IgG and one IgM isotype, were isolated. The properties of IgG1/κ monoclonal (designated as Mab-PAR-1) was studied further. Mab-PAR-1 was shown to be functional in western blot against denatured recombinant protein and viral particles. In immunofluorescence assay, the antibody tested positive for nineteen PeV-A1 isolates while showing no cross-reactivity to fourteen entero- and rhinovirus types. In addition, Mab-PAR-1 showed positive reactivity against five other cultivable parechovirus types 2-6. A unique Mab-PAR-1 epitope located in the junction of the three capsid proteins VP0, VP1, and VP3 was identified using a peptide library screen. This study demonstrates that PeV-A1-VP0 protein is functional antigen for developing monoclonal antibody for diagnosis of broad range of parechovirus infections.

Preperitoneal Fat Grafting Inhibits the Formation of Intra-abdominal Adhesions in Mice.

BACKGROUND: Adhesion formation contributes to postoperative complications in abdominal and gynaecological surgery. Thus far, the prevention and treatment strategies have focused on mechanical barriers in solid and liquid form, but these methods are not in routine use. As autologous fat grafting has become popular in treatment of hypertrophic scars because of its immunomodulatory effects, we postulated that fat grafting could also prevent peritoneal adhesion through similar mechanisms. METHODS: This was a control versus intervention study to evaluate the effect of fat grafting in the prevention on peritoneal adhesion formation. An experimental mouse model for moderate and extensive peritoneal adhesions was used (n = 4-6 mice/group). Adhesions were induced mechanically, and a free epididymal fat graft from wild type or CAG-DsRed mice was injected preperitoneally immediately after adhesion induction. PET/CT imaging and scaling of the adhesions were performed, and samples were taken for further analysis at 7 and 30 days postoperation. Macrophage phenotyping was further performed from peritoneal lavage samples, and the expression of inflammatory cytokines and mesothelial layer recovery were analysed from peritoneal tissue samples. RESULTS: Fat grafting significantly inhibited the formation of adhesions. PET/CT results did not show prolonged inflammation in any of the groups. While the expression of anti-inflammatory and anti-fibrotic IL-10 was significantly increased in the peritoneum of the fat graft-treated group at 7 days, tissue-resident and repairing M2 macrophages could no longer be detected in the fat graft at this time point. The percentage of the continuous, healed peritoneum as shown by Keratin 8 staining was greater in the fat graft-treated group after 7 days. CONCLUSIONS: Fat grafting can inhibit the formation of peritoneal adhesions in mice. Our results suggest that fat grafting promotes the peritoneal healing process in a paracrine manner thereby enabling rapid regeneration of the peritoneal mesothelial cell layer.

Genome Sequences of RIGVIR Oncolytic Virotherapy Virus and Five Other Echovirus 7 Isolates.

We report here the nearly complete Illumina-sequenced consensus genome sequences of six isolates of echovirus 7 (E7), including oncolytic virotherapy virus RIGVIR and the Wallace prototype. Amino acid identities within the coding region were highly conserved across all isolates, ranging from 95.31% to 99.73%.

Integrins are not essential for entry of coxsackievirus A9 into SW480 human colon adenocarcinoma cells.

BACKGROUND: Coxsackievirus A9 (CV-A9) is a pathogenic enterovirus type within the family Picornaviridae. CV-A9 infects A549 human epithelial lung carcinoma cells by attaching to the αVß6 integrin receptor through a highly conserved Arg-Gly-Asp (RGD) motif, which is located at the exposed carboxy-terminus of the capsid protein VP1 detected in all studied clinical isolates. However, genetically-modified CV-A9 that lacks the RGD motif (CV-A9-RGDdel) has been shown to be infectious in some cell lines but not in A549, suggesting that RGD-mediated integrin binding is not always essential for efficient entry of CV-A9. METHODS: Two cell lines, A549 and SW480, were used in the study. SW480 was the study object for the integrin-independent entry and A549 was used as the control for integrin-dependent entry. Receptor levels were quantitated by cell sorting and quantitative PCR. Antibody blocking assay and siRNA silencing of receptor-encoding genes were used to block virus infection. Peptide phage display library was used to identify peptide binders to CV-A9. Immunofluorescence and confocal microscopy were used to visualize the virus infection in the cells. RESULTS: We investigated the receptor use and early stages of CV-A9 internalization to SW480 human epithelial colon adenocarcinoma cells. Contrary to A549 infection, we showed that both CV-A9 and CV-A9-RGDdel internalized into SW480 cells and that function-blocking anti-αV integrin antibodies had no effect on the binding and entry of CV-A9. Whereas siRNA silencing of ß6 integrin subunit had no influence on virus infection in SW480, silencing of ß2-microglobulin (ß2M) inhibited the virus infection in both cell lines. By using a peptide phage display screening, the virus-binding peptide identical to the N-terminal sequence of HSPA5 protein was identified and shown to block the virus infection in both A549 and SW480 cell lines. HSPA5 was also found to co-localize with CV-A9 at the SW480 cell periphery during the early stages of infection by confocal microscopy. CONCLUSIONS: The data suggest that while αVß6 integrin is essential for CV-A9 in A549 cell line, it is not required in SW480 cell line in which ß2M and HSPA5 alone are sufficient for CV-A9 infection. This suggests that the choice of CV-A9 receptor(s) is dependent on the tissue/cellular environment.

Human Parechovirus 1 Infection Occurs via αVß1 Integrin.

Human parechovirus 1 (HPeV-1) (family Picornaviridae) is a global cause of pediatric respiratory and CNS infections for which there is no treatment. Although biochemical and in vitro studies have suggested that HPeV-1 binds to αVß1, αVß3 and αVß6 integrin receptor(s), the actual cellular receptors required for infectious entry of HPeV-1 remain unknown. In this paper we analyzed the expression profiles of αVß1, αVß3, αVß6 and α5ß1 in susceptible cell lines (A549, HeLa and SW480) to identify which integrin receptors support HPeV-1 internalization and/or replication cycle. We demonstrate by antibody blocking assay, immunofluorescence microscopy and RT-qPCR that HPeV-1 internalizes and replicates in cell lines that express αVß1 integrin but not αVß3 or αVß6 integrins. To further study the role of ß1 integrin, we used a mouse cell line, GE11-KO, which is deficient in ß1 expression, and its derivate GE11-ß1 in which human integrin ß1 subunit is overexpressed. HPeV-1 (Harris strain) and three clinical HPeV-1 isolates did not internalize into GE11-KO whereas GE11-ß1 supported the internalization process. An integrin ß1-activating antibody, TS2/16, enhanced HPeV-1 infectivity, but infection occurred in the absence of visible receptor clustering. HPeV-1 also co-localized with ß1 integrin on the cell surface, and HPeV-1 and ß1 integrin co-endocytosed into the cells. In conclusion, our results demonstrate that in some cell lines the cellular entry of HPeV-1 is primarily mediated by the active form of αVß1 integrin without visible receptor clustering.

Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates.

Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype) strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54) CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9) had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%), heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin) completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.

Genome Sequence of Coxsackievirus A6, Isolated during a Hand-Foot-and-Mouth Disease Outbreak in Finland in 2008.

Reports of hand-foot-and-mouth disease (HFMD) outbreaks caused by coxsackievirus A6 have increased worldwide after the report of the first outbreak in Finland in 2008. The complete genome of the first outbreak strain from a vesicle fluid specimen was determined.

Endocytosis of integrin-binding human picornaviruses.

Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9), echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1) has no RGD and uses integrin α2ß1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive ß1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.