Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor ␣2␤1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, ␣2␤1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.

Thromboembolism frequently occurs during acute lymphoblastic leukemia (ALL) therapy. We prospectively registered thromboembolic events during the treatment of 1772 consecutive Nordic/Baltic patients with ALL aged 1 to 45 years who were treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol (July 2008-April 2017). The 2.5-year cumulative incidence of thromboembolism (N = 137) was 7.9% (95% confidence interval [CI], 6.6-9.1); it was higher in patients aged at least 10 years ( < .0001). Adjusted hazard ratios (HRas) were associated with greater age (range, 10.0-17.9 years: HRa, 4.9 [95% CI, 3.1-7.8; < .0001]; 18.0-45.9 years: HRa, 6.06 [95% CI, 3.65-10.1; < .0001]) and mediastinal mass at ALL diagnosis (HRa, 2.1; 95% CI, 1.0-4.3; = .04). In a multiple absolute risk regression model addressing 3 thromboembolism risk factors, age at least 10 years had the largest absolute risk ratio (RR, 4.7 [95% CI, 3.1-7.1]; RR, 2.0 [95% CI, 1.2-3.1]; RR, 1.6 [95% CI, 1.0-2.6]). Patients aged 18.0 to 45.9 years had an increased hazard of pulmonary embolism (HRa, 11.6; 95% CI, 4.02-33.7; < .0001), and patients aged 10.0 to 17.9 years had an increased hazard of cerebral sinus venous thrombosis (HRa, 3.3; 95% CI, 1.5-7.3; = .003) compared with children younger than 10.0 years. Asparaginase was truncated in 38/128 patients with thromboembolism, whereas thromboembolism diagnosis was unassociated with increased hazard of relapse ( = .6). Five deaths were attributable to thromboembolism, and patients younger than 18.0 years with thromboembolism had increased hazard of dying compared with same-aged patients without thromboembolism (both ≤ .01). In conclusion, patients aged at least 10 years could be candidates for preemptive antithrombotic prophylaxis. However, the predictive value of age 10 years or older, enlarged lymph nodes, and mediastinal mass remain to be validated in another cohort.

Integrin ␣ 2 subunit forms in the complex with the ␤ 1 subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special "inserted" domain (I domain) in the integrin ␣ 2 subunit. Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet ␣ 2 ␤ 1 integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant r␣ 2 I domain to type I collagen led us to study jararhagin further. Synthetic peptides representing hydrophilic and charged sequences of jararhagin, including the RSECD sequence replacing the well known RGD motif in the disintegrinlike domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit r␣ 2 I domain binding to collagen, a basic peptide from the metalloproteinase domain proved to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind strongly to human recombinant ␣ 2 I domain and to prevent its binding to type I and IV collagens and to laminin-1. Mutational analysis indicated that a sequence of three amino acids, arginine-lysinelysine (RKK), is essential for r␣ 2 I domain binding, whereas the mutation of the other amino acids in the peptide had little if any effect on its binding function. Importantly, the peptide was functional only in the cyclic conformation and its affinity was strictly dependent on the size of the cysteine-constrained loop. Furthermore, the peptide could not bind to ␣ 2 I domain in the absence of Mg 2؉ , suggesting that the conformation of the I domain was critical, as well. Cells could attach to the peptide only if they expressed ␣ 2 ␤ 1 integrin, and the attachment was inhibited by anti-integrin antibodies.Integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 are the major cellular receptors for native collagens (for review, see Refs. 1 and 2). Like all integrins their interaction with ligands is dependent on divalent cations (3). The ␣ 1 and ␣ 2 subunits contain an special inserted domain, the I domain, resembling the A domain found e.g. in von Willenbrand factor (4). It is evident that ␣ 1 I and ␣ 2 I domains are responsible for the primary recognition of collagen by the corresponding integrins (5, 6). Two other ligands for ␣ 2 ␤ 1 integrin, namely laminin-1 and echovirus-1, each bind to ␣ 2 I domain, as well. However, echovirus-1 seems to recognize a different site on the ␣ 2 I domain than the matrix proteins do (7).The binding sites of ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins in collagens have been localized to the triple helical areas of the molecules (8, 9). One peptide sequence derived from the collagen ␣ chain has been reported to block integrin-collagen interaction (10), but in many studies it has been ineffective and it probably does not represent the actual binding site in collagen (11-13). More likely, collagen-receptor integrins recognize amino acid resi...

In order to determine the overall molecular heterogeneity of echoviruses (EVs) we performed a genetic analysis of the prototype strains. Nucleotide and derived amino acid sequences from different genomic regions (5'UTR, capsid protein-coding and 3D polymerase genes) were used for molecular comparisons. On the basis of a comparison of partial amino acid sequences from the capsid protein VP2, all the sequenced EVs excluding EV22 and EV23 form a single cluster which is genetically homogeneous. All previously sequenced coxsackie B viruses (CBVs) and coxsackievirus A9 also belong to this same genetic cluster. Similar results were obtained when the 5'UTR or 3D polymerase gene sequences were used in comparisons. When amino acid sequences of the major capsid proteins of EV1 and EV16 were compared to those of previously sequenced enteroviruses, the length of the loops connecting the flsheets appeared to be relatively constant in the EV/CBV cluster. It can be concluded that EVs and CBVs have diverged relatively late in evolution.

We have previously shown that echovirus 1 (EV1) infection increases the mRNA levels of cellular immediate-early (IE) genes in host cells. Here we provide further evidence that the induction of junB, c-jun, and c-fos genes is due to active viral macromolecular synthesis rather than to the interaction of EV1 with its receptor, alpha2beta1 integrin. Nuclear run-on transcription assays indicated that differences in mRNA levels in infected and uninfected cells are brought about by regulation at the transcriptional level. EV1 infection induced the phosphorylation of both the stress-related p38 mitogen-activated protein kinase (MAPK) and the growth signal-related ERK1/2 MAPKs. Studies with selective MAPK inhibitors revealed that p38 was the main inducer of junB expression, whereas both MAPK pathways were involved in the induction of c-fos. Activation of AP-1 genes was also observed to occur during infections with other enteroviruses and with Semliki Forest A7(74) virus, suggesting that the phosphorylation of MAPKs and induction of AP-1 gene expression may be important regulators of host cell behavior during viral infections.

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We conclude that early lymphocyte subset counts following allogeneic HSCT have an association with aGVHD and post transplant outcome.