Identification of protease and ADP-ribose 1''-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3.

The replicase polyproteins, pp1a and pp1ab, of porcine Transmissible gastroenteritis virus (TGEV) have been predicted to be cleaved by viral proteases into 16 non-structural proteins (nsp). Here, enzymic activities residing in the amino-proximal region of nsp3, the largest TGEV replicase processing product, were characterized. It was shown, by in vitro translation experiments and protein sequencing, that the papain-like protease 1, PL1pro, but not a mutant derivative containing a substitution of the presumed active-site nucleophile, Cys1093, cleaves the nsp2|nsp3 site at 879Gly|Gly880. By using an antiserum raised against the pp1a/pp1ab residues 526–713, the upstream processing product, nsp2, was identified as an 85 kDa protein in TGEV-infected cells. Furthermore, PL1pro was confirmed to be flanked at its C terminus by a domain (called X) that mediates ADP-ribose 1''-phosphatase activity. Expression and characterization of a range of bacterially expressed forms of this enzyme suggest that the active X domain comprises pp1a/pp1ab residues Asp1320–Ser1486.

Development and clinical validation of multiplex TaqMan® assays for rapid diagnosis of viral gastroenteritis.

There is a need to provide rapid, sensitive, and often high throughput detection of pathogens in diagnostic virology. Viral gastroenteritis is a serious health issue often leading to hospitalization in the young, the immunocompromised and the elderly. The common causes of viral gastroenteritis include rotavirus, norovirus (genogroups I and II), astrovirus, and group F adenoviruses (serotypes 40 and 41). This article describes the work-up of two internally controlled multiplex, probe-based PCR assays and reports on the clinical validation over a 3-year period, March 2007 to February 2010. Multiplex assays were developed using a combination of TaqMan™ and minor groove binder (MGB™) hydrolysis probes. The assays were validated using a panel of 137 specimens, previously positive via a nested gel-based assay. The assays had improved sensitivity for adenovirus, rotavirus, and norovirus (97.3% vs. 86.1%, 100% vs. 87.8%, and 95.1% vs. 79.5%, respectively) and also more specific for targets adenovirus, rotavirus, and norovirus (99% vs. 95.2%, 100% vs. 93.6%, and 97.9% vs. 92.3%, respectively). For the specimens tested, both assays had equal sensitivity and specificity for astrovirus (100%). Overall the probe-based assays detected 16 more positive specimens than the nested gel-based assay. Post-introduction to the routine diagnostic service, a total of 9,846 specimens were processed with multiplex 1 and 2 (7,053 pediatric, 2,793 adult) over the 3-year study period. This clinically validated, probe-based multiplex testing algorithm allows highly sensitive and timely diagnosis of the four most prominent causes of viral gastroenteritis.

Association of the G4 rotavirus genotype with gastroenteritis in adults

Rotavirus is the most common etiological cause of acute viral gastroenteritis in infants and young children worldwide, yet its role in the adult population is less well understood. We have recently identified rotavirus as the causative agent of severe diarrhea in adults, specifically in two gastroenteritis outbreaks in separate care for the elderly homes. Strain typing has shown the continued presence of P[8]G1, the emergence of P[8]G9, and the reemergence of P[8]G4. A total of 26 community cases and 6 outbreak cases of rotavirus infection, positive via a molecular screening assay, were subsequently amplified using VP4 and VP7 specific primers (Con2/Con3 and 1A/1B primer sets, respectively). The age range of patients investigated was from

Serological correlate of protection against norovirus-induced gastroenteritis

Norovirus infection is the leading cause of acute nonbacterial gastroenteritis. Histoblood group antigens (HBGAs) are host susceptibility determinants for Norwalk virus (NV) infection. We hypothesized that antibodies that block NV-HBGA binding are associated with protection from clinical illness following NV exposure.

The effects of climate change on ovine parasitic gastroenteritis determined using veterinary surveillance and meteorological data for Northern Ireland over the period 1999-2009

While the influence of temperature and moisture on the free-living stages of gastrointestinal nematodes have been described in detail, and evidence for global climate change is mounting, there have been only a few attempts to relate altered incidence or seasonal patterns of disease to climate change. Studies of this type have been completed for England Scotland and Wales, but not for Northern Ireland (NI). Here we present an analysis of veterinary diagnostic data that relates three categories of gastrointestinal nematode infection in sheep to historical meteorological data for NI. The infections are: trichostrongylosis/teladorsagiosis (Teladorsagia/Trichostrongylus), strongyloidosis and nematodirosis. This study aims to provide a baseline for future climate change analyses and to provide basic information for the development of nematode control programmes. After identifying and evaluating possible sources of bias, climate change was found to be the most likely explanation for the observed patterns of change in parasite epidemiology, although other hypotheses could not be refuted. Seasonal rates of diagnosis showed a uniform year-round distribution for Teladorsagia and Trichostrongylus infections, suggesting consistent levels of larval survival throughout the year and extension of the traditionally expected seasonal transmission windows. Nematodirosis showed a higher level of autumn than Spring infection, suggesting that suitable conditions for egg and larval development occurred after the Spring infection period. Differences between regions within the Province were shown for strongyloidosis, with peaks of infection falling in the period September-November. For all three-infection categories (trichostrongylosis/teladorsagiosis, strongyloidosis and nematodirosis), significant differences in the rates of diagnosis, and in the seasonality of disease, were identified between regions. (C) 2012 Elsevier B.V. All rights reserved.

Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs

A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. We determined crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and we constructed a homology model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Molecular modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.

Conservation of substrate specificities among coronavirus main proteases.

The key enzyme in coronavirus replicase polyprotein processing is the coronavirus main protease, 3CL(pro). The substrate specificities of five coronavirus main proteases, including the prototypic enzymes from the coronavirus groups I, II and III, were characterized. Recombinant main proteases of human coronavirus (HCoV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus, avian infectious bronchitis virus and mouse hepatitis virus (MHV) were tested in peptide-based trans-cleavage assays. The determination of relative rate constants for a set of corresponding HCoV, TGEV and MHV 3CL(pro) cleavage sites revealed a conserved ranking of these sites. Furthermore, a synthetic peptide representing the N-terminal HCoV 3CL(pro) cleavage site was shown to be effectively hydrolysed by noncognate main proteases. The data show that the differential cleavage kinetics of sites within pp1a/pp1ab are a conserved feature of coronavirus main proteases and lead us to predict similar processing kinetics for the replicase polyproteins of all coronaviruses.

Neutral Genomic Microevolution of a Recently Emerged Pathogen, Salmonella enterica Serovar Agona

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks. © 2013 Achtman et al

Atmospheric pressure, nonthermal plasma inactivation of MS2 bacteriophage: effect of oxygen concentration on virucidal activity

AimsThe main aim of this study was to determine the virucidal inactivation efficacy of an in-house-designed atmospheric pressure, nonthermal plasma jet operated at varying helium/oxygen feed gas concentrations against MS2 bacteriophage, widely employed as a convenient surrogate for human norovirus.

Methods and ResultsThe effect of variation of percentage oxygen concentration in the helium (He) carrier gas was studied and found to positively correlate with MS2 inactivation rate, indicating a role for reactive oxygen species (ROS) in viral inactivation. The inactivation rate constant increased with increasing oxygen concentrations up to 075% O-2. 3 log(10) (999%) reductions in MS2 viability were achieved after 3min of exposure to the plasma source operated in a helium/oxygen (9925%:075%) gas mixture, with >7 log(10) reduction after 9min exposure.

ConclusionsAtmospheric pressure, nonthermal plasmas may have utility in the rapid disinfection of virally contaminated surfaces for infection control applications.

Significance and Impact of StudyThe atmospheric pressure, nonthermal plasma jet employed in this study exhibits rapid virucidal activity against a norovirus surrogate virus, the MS2 bacteriophage, which is superior to previously published inactivation rates for chemical disinfectants.

Community-acquired infections and their association with myeloid malignancies

Introduction: Antigenic stimulation is a proposed aetiologic mechanism for many haematological malignancies. Limited evidence suggests that community-acquired infections may increase the risk of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). However, associations with other myeloid malignancies including chronic myeloid leukaemia (CML) and myeloproliferative neoplasms (MPNs) are unknown.

Materials and methods: Using the Surveillance, Epidemiology and End Result (SEER)-Medicare database, fourteen community-acquired infections were compared between myeloid malignancy patients [AML (n=8489), CML (n=3626) diagnosed 1992-2005; MDS (n=3072) and MPNs (n=2001) diagnosed 2001-2005; and controls (200,000 for AML/CML and 97,681 for MDS/MPN]. Odds ratios (ORs) and 95% confidence intervals were adjusted for gender, age and year of selection excluding infections diagnosed in the 13-month period prior to selection to reduce reverse causality.

Results: Risk of AML and MDS respectively, were significantly associated with respiratory tract infections, bronchitis (ORs 1.20 [95% CI: 1.14-1.26], 1.25 [95% CI: 1.16-1.36]), influenza (ORs 1.16 [95% CI: 1.07-1.25], 1.29 [95% CI: 1.16-1.44]), pharyngitis (ORs 1.13 [95% CI: 1.06-1.21], 1.22 [95% CI: 1.11-1.35]), pneumonia (ORs 1.28 [95% CI: 1.21-1.36], 1.52 [95% CI: 1.40-1.66]), sinusitis (ORs 1.23 [95% CI: 1.16-1.30], 1.25 [95% CI: 1.15-1.36]) as was cystitis (ORs 1.13 [95% CI: 1.07-1.18], 1.26 [95% CI: 1.17-1.36]). Cellulitis (OR 1.51 [95% CI: 1.39-1.64]), herpes zoster (OR 1.31 [95% CI: 1.14-1.50]) and gastroenteritis (OR 1.38 [95% CI: 1.17-1.64]) were more common in MDS patients than controls. For CML, associations were limited to bronchitis (OR 1.21 [95% CI: 1.12-1.31]), pneumonia (OR 1.49 [95% CI: 1.37-1.62]), sinusitis (OR 1.19 [95% CI: 1.09-1.29]) and cellulitis (OR 1.43 [95% CI: 1.32-1.55]) following Bonferroni correction. Only cellulitis (OR 1.34 [95% CI: 1.21-1.49]) remained significant in MPN patients. Many infections remained elevated when more than 6 years of preceding claims data were excluded.

Discussion: Common community-acquired infections may be important in the malignant transformation of the myeloid lineage. Differences in the aetiology of classic MPNs and other myeloid malignancies require further exploration.