Human enteroviruses (EVs) and more recently parechoviruses (HPeVs) have been identified as the principal viral causes of neonatal sepsis-like disease and meningitis. The relative frequencies of specific EV and HPeV types were determined over a 5-year surveillance period using highly sensitive EV and HPeV PCR assays for screening 4,168 cerebrospinal fluid (CSF) specimens collected from hospitalized individuals between 2005 and 2010 in Edinburgh. Positive CSF samples were typed by sequencing of VP1. From the 201 EV and 31 HPeV positive (uncultured) CSF samples on screening, a high proportion of available samples could be directly typed (176/182, 97%). Highest frequencies of EV infections occurred in young adults (n = 43; 8.6%) although a remarkably high proportion of positive samples (n = 98; 46%) were obtained from young infants (<3 months). HPeV infections were seen exclusively in children under the age of 3 months (31/1,105; 2.8%), and confined to spring on even-numbered years (22% in March 2006, 25% in April 2008, and 22% in March 2010). In contrast, EV infections were distributed widely across the years. Twenty different EV serotypes were detected; E9, E6, and CAV9 being found most frequently, whereas all but one HPeVs were type 3. Over this period, HPeV3 was identified as the most prevalent picornavirus type in CNS-related infections with similarly high incidences of EV infection frequencies in very young children. The highly sensitive virus typing methods applied in this study will assist further EV and HPeV screening of sepsis and meningitis cases as well as in future molecular epidemiological studies and population surveillance.

The recent Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola and Zika virus outbreaks exemplify the continued threat of (re-)emerging viruses to human health, and our inability to rapidly develop effective therapeutic countermeasures. Many viruses, including MERS-CoV and the Crimean-Congo hemorrhagic fever virus (CCHFV) encode deubiquitinating (DUB) enzymes that are critical for viral replication and pathogenicity. They bind and remove ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) from cellular proteins to suppress host antiviral innate immune responses. A variety of viral DUBs (vDUBs), including the MERS-CoV papain-like protease, are responsible for cleaving the viral replicase polyproteins during replication, and are thereby critical components of the viral replication cycle. Together, this makes vDUBs highly attractive antiviral drug targets. However, structural similarity between the catalytic cores of vDUBs and human DUBs complicates the development of selective small molecule vDUB inhibitors. We have thus developed an alternative strategy to target the vDUB activity through a rational protein design approach. Here, we report the use of phage-displayed ubiquitin variant (UbV) libraries to rapidly identify potent and highly selective protein-based inhibitors targeting the DUB domains of MERS-CoV and CCHFV. UbVs bound the vDUBs with high affinity and specificity to inhibit deubiquitination, deISGylation and in the case of MERS-CoV also viral replicative polyprotein processing. Co-crystallization studies further revealed critical molecular interactions between UbVs and MERS-CoV or CCHFV vDUBs, accounting for the observed binding specificity and high affinity. Finally, expression of UbVs during MERS-CoV infection reduced infectious progeny titers by more than four orders of magnitude, demonstrating the remarkable potency of UbVs as antiviral agents. Our results thereby establish a strategy to produce protein-based inhibitors that could protect against a diverse range of viruses by providing UbVs via mRNA or protein delivery technologies or through transgenic techniques.

Human rhinoviruses (HRVs) can be divided into three species; HRV-A to HRV-C. Up to 148 different HRV (sero)types have been identified to date. Because of sequence similarity between 5'-NCR of HRVs and enteroviruses (EVs), it is problematic to design EV-specific RT-PCR assays. The aims of this study were to assess the rate of false-detection of different rhinoviruses by EV RT-PCR, and to evaluate the diagnostic and clinical significance of such cross-reactivity. In vitro RNA transcripts of HRV A-C created from cDNA templates were quantified spectrophotometrically. Six hundred twenty-one stool samples screened as part of routine diagnostic for EV, 17 EV-positive stool samples referred for typing, 288 stool samples submitted for gastroenteritis investigations, and 1,500 CSF samples were included in the study. EV-specific RT-PCR detected RNA transcripts of HRV-A1b, HRV-B14, and HRV-Crpat18 but with 10-1,000 reduced sensitivity compared to EV transcripts. Screening fecal samples by EV RT-PCR identified 13 positive samples identified subsequently as rhinoviruses; a further 26 HRV-positive samples were identified by nested HRV RT-PCR. All individuals were hospitalized and presented mostly with diarrhea. A total of 26 HRV types were identified (HRV-A: 46%; HRV-B: 13%; HRV-C: 41%). Results confirm that EV-specific RT-PCR can detect HRVs, and at a practical level, identify potential problems of interpretation if fecal samples are used for surrogate screening in cases of suspected viral meningitis. High detection frequencies (10%) and viral loads in stool samples provide evidence for enteric replication of HRV, and its association with enteric disease requires further etiological studies.

bCoxsackievirus A9 (CAV9), a member of the Picornaviridae family, uses an RGD motif in the VP1 capsid protein to bind to integrin ␣v␤6 during cell entry. Here we report that two CAV9 isolates can bind to the heparan sulfate/heparin class of proteoglycans (HSPG). Sequence analysis identified an arginine (R) at position 132 in VP1 in these two isolates, rather than a threonine (T) as seen in the nonbinding strains tested. We introduced a T132R substitution into the HSPG-nonbinding strain Griggs and recovered infectious virus capable of binding to immobilized heparin, unlike the parental Griggs strain. The known CAV9 structure was used to identify the location of VP1 position 132, 5 copies of which were found to cluster around the 5-fold axis of symmetry, presumably producing a region of positive charge which can interact with the negatively charged HSPG. Analysis of several enteroviruses of the same species as CAV9, Human enterovirus B (HEV-B), identified examples from 5 types in which blocking of infection by heparin was coincident with an arginine (or another basic amino acid, lysine) at a position corresponding to 132 in VP1 in CAV9. Together, these data show that membrane-associated HSPG can serve as a (co)receptor for some CAV9 and other HEV-B strains and identify symmetry-related clustering of positive charges as one mechanism by which HSPG binding can be achieved. This is a potentially powerful mechanism by which a single amino acid change could generate novel receptor binding capabilities, underscoring the plasticity of host-cell interactions in enteroviruses.