Repeated examination of natural sapovirus infections in pig litters raised under experimental conditions.

BACKGROUND: Porcine sapovirus, belonging to the family Caliciviridae, is an enteric virus that is widespread in the swine industry worldwide. A total of 14 sapovirus genogroups have been suggested and the most commonly found genogroup in swine is genogroup III (GIII). The goal of the present experiment was to examine the presence of sapovirus in 51 naturally infected pigs at two different time points. The pigs were kept under experimental conditions after weaning. Previous studies on sapovirus have primarily been of a cross sectional nature, typically prevalence studies performed on farms and abattoirs. In the present study, faecal samples, collected from each pig at 5½ weeks and 15-18 weeks of age, were analysed for sapovirus by reverse transciptase polymerase chain reaction and positive findings were genotyped by sequencing. RESULTS: At 5½ weeks of age, sapovirus was detected in the majority of the pigs. Sequencing revealed four different strains in the 5½ week olds-belonging to genogroups GIII and GVII. Ten to 13 weeks later, the virus was no longer detectable from stools of infected pigs. However, at this time point 13 pigs were infected with another GIII sapovirus strain not previously detected in the pigs studied. This GIII strain was only found in pigs that, in the initial samples, were virus-negative or positive for GVII. CONCLUSIONS: At 5 weeks of age 74 % of the pigs were infected with sapovirus. At 15-18 weeks of age all pigs had cleared their initial infection, but a new sapovirus GIII strain was detected in 25 % of the pigs. None of the pigs initially infected with the first GIII strain were reinfected with this new GIII strain, which may indicate the presence of a genogroup-specific immunity.

Identification of immunogenic and virulence-associated Campylobacter jejuni proteins.

With the aim of identifying proteins important for host interaction and virulence, we have screened an expression library of NCTC 11168 Campylobacter jejuni genes for highly immunogenic proteins. A commercial C. jejuni open reading frame (ORF) library consisting of more than 1,600 genes was transformed into the Escherichia coli expression strain BL21(DE3), resulting in 2,304 clones. This library was subsequently screened for immunogenic proteins using antibodies raised in rabbit against a clinical isolate of C. jejuni; this resulted in 52 highly reactive clones representing 25 different genes after sequencing. Selected candidate genes were inactivated in C. jejuni NCTC 11168, and the virulence was examined using INT 407 epithelial cell line and motility, biofilm, autoagglutination, and serum resistance assays. These investigations revealed C. jejuni antigen 0034c (Cj0034c) to be a novel virulence factor and support the usefulness of the method. Further, several antigens were tested as vaccine candidates in two mouse models, in which Cj0034c, Cj0404, and Cj0525c resulted in a reduction of invasion in spleen and liver after challenge.

Distinct features of circulating microparticles and their relationship to clinical manifestations in systemic lupus erythematosus.

OBJECTIVE: Characterization of the abundance, origin, and annexin V (AnxV)-binding capabilities of circulating microparticles (MPs) in SLE patients and healthy controls and to determine any associations with clinical parameters. METHODS: Seventy unselected SLE patients and 29 sex- and age-matched healthy control subjects were included in the study. MPs were isolated from citrate-treated plasma and characterized by flow cytometry using AnxV or antibodies to platelet, leukocyte, or endothelial cell surface markers. RESULTS: SLE patients had significantly increased concentrations of AnxV-nonbinding (AnxV-) MPs (P<0.0001), while the concentrations of total MPs (P=0.011) and AnxV-binding (AnxV+) MPs (P<0.0001) were decreased, as compared with controls. Based on flow cytometric characteristics, 2 subgroups of AnxV- MPs could be discerned: AnxV- cell-derived MPs (CDMPs) and AnxV- MPs of unknown nature (UNMPs). Both fractions were significantly increased in SLE patients (P=0.007 and P=0.0018, respectively). Platelet- and leukocyte-derived MPs were decreased in the SLE patients (P<0.0001), whereas no difference was observed for endothelial cell-derived MPs (P=0.14). The concentrations of AnxV- CDMPs correlated with the concentrations of endothelial cell-derived MPs, the disease activity score, active nephritis, hypertension, history of arterial thrombosis, and triglyceride levels (P<0.05 for all comparisons). CONCLUSION: The concentrations and composition of MPs in SLE patients differ markedly from those in healthy subjects. Overall MP numbers were significantly decreased, but two distinct subpopulations of AnxV- MPs were significantly increased. These findings call for further characterization of MPs in SLE patients to elucidate their role in disease pathogenesis.

Glycosaminoglycans enhance the fibrillation propensity of the ß2-microglobulin cleavage variant--ΔK58-ß2m.

Dialysis related amyloidosis (DRA) is a serious complication to long-term hemodialysis treatment which causes clinical symptoms such as carpal tunnel syndrome and destructive arthropathies. The disease is characterized by the assembly and deposition of ß2-microglobulin (ß2m) predominantly in the musculoskeletal system, but the initiating events leading to ß2m amyloidogenesis and the molecular mechanisms underlying amyloid fibril formation are still unclear. Glycosaminoglycans (GAGs) and metal ions have been shown to be related to the onset of protein aggregation and to promote de novo fiber formation. In this study, we show that fibrillogenesis of a cleavage variant of ß2m, ΔK58-ß2m, which can be found in the circulation of hemodialysis patients and is able to fibrillate at near-physiological pH in vitro, is affected by the presence of copper ions and heparan sulfate. It is found that the fibrils generated when heparan sulfate is present have increased length and diameter, and possess enhanced stability and seeding properties. However, when copper ions are present the fibrils are short, thin and less stable, and form at a slower rate. We suggest that heparan sulfate stabilizes the cleaved monomers in the early aggregates, hereby promoting the assembly of these into fibrils, whereas the copper ions appear to have a destabilizing effect on the monomers. This keeps them in a structure forming amorphous aggregates for a longer period of time, leading to the formation of spherical bodies followed by the assembly of fibrils. Hence, the in vivo formation of amyloid fibrils in DRA could be initiated by the generation of ΔK58-ß2m which spontaneously aggregate and form fibrils. The fibrillogenesis is enhanced by the involvement of GAGs and/or metal ions, and results in amyloid-like fibrils able to promote the de novo formation of ß2m amyloid by a scaffold mechanism.

Incidence, diversity, and molecular epidemiology of sapoviruses in swine across Europe.

Porcine sapovirus is an enteric calicivirus in domestic pigs that belongs to the family Caliciviridae. Some porcine sapoviruses are genetically related to human caliciviruses, which has raised public health concerns over animal reservoirs and the potential cross-species transmission of sapoviruses. We report on the incidence, genetic diversity, and molecular epidemiology of sapoviruses detected in domestic pigs in a comprehensive study conducted in six European countries (Denmark, Finland, Hungary, Italy, Slovenia, and Spain) between 2004 and 2007. A total of 1,050 swine fecal samples from 88 pig farms were collected and tested by reverse transcription-PCR for sapoviruses, and positive findings were confirmed by sequencing. Sapoviruses were detected in 80 (7.6%) samples collected on 39 (44.3%) farms and in every country. The highest prevalence was seen among piglets aged 2 to 8 weeks, and there was no significant difference in the proportion of sapovirus-positive findings for healthy animals and animals with diarrhea in Spain and Denmark (the only countries where both healthy animals and animals with diarrhea were tested). On the basis of the sequence of the RNA polymerase region, highly heterogeneous populations of viruses representing six different genogroups (genogroups III, VI, VII, and VIII, including potential new genogroups IX and X) were identified, with a predominance of genogroup GIII (50.6%). Genogroup VIII, found in five of the six countries, had the highest degree of homology (up to 66% at the amino acid level) to human sapovirus strains. Sapoviruses are commonly circulating and endemic agents in swine herds throughout Europe. Highly heterogeneous and potential new genogroups of sapoviruses were found in pigs; however, no "human-like" sapoviruses were detected.

Genetic diversity of sapovirus infections in Danish children 2005-2007.

BACKGROUND: Sapoviruses are known to cause gastroenteritis mainly in young children. OBJECTIVES: To establish a collection of sapoviruses and to gain knowledge about the genetic diversity and epidemiology of sapoviruses circulating in children in Denmark. STUDY DESIGN: During a 24-month period in 2005-2007 samples from 1104 children, aged 0-3 years, submitted for acute gastroenteritis diagnostics, were analysed for sapoviruses by real-time PCR. Genotyping of positive findings was carried out by sequencing part of the capsid gene, and in several cases also part of the polymerase gene. RESULTS: Sapoviruses were detected in stool samples from 97 children (9%), with the highest prevalence in the 7-18 months age group. Viruses from three genogroups and seven genotypes were found. Genotype I.1 was demonstrated in half of the positive samples and was observed throughout the study period. The less common types seemed to appear during shorter periods, often in succession of each other. Positive samples were detected throughout the study period. The only months, in both years studied, with high proportions of positive samples were September, November and February. CONCLUSIONS: Sapoviruses were commonly found in children with gastroenteritis in Denmark. No clear seasonal pattern could be seen. Genotype I.1 was clearly the most common genotype found, but several other genotypes circulated during shorter periods.

A beta2-microglobulin cleavage variant fibrillates at near-physiological pH.

Beta2-microglobulin (beta2m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of beta2m are still largely unknown. In vitro, acidic conditions, pH < 4.5, induce amyloid fibrillation of native beta2m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of beta2m--found in the circulation of many dialysis patients--is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native beta2m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of beta2m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native beta2m. This may explain the apparent lack of detectable amounts of the variant beta2m in extracts of amyloid from DRA patients.

Emergence of new norovirus variants on spring cruise ships and prediction of winter epidemics.

In June 2006, reported outbreaks of norovirus on cruise ships suddenly increased; 43 outbreaks occurred on 13 vessels. All outbreaks investigated manifested person-to-person transmission. Detection of a point source was impossible because of limited investigation of initial outbreaks and data sharing. The most probable explanation for these outbreaks is increased norovirus activity in the community, which coincided with the emergence of 2 new GGII.4 variant strains in Europe and the Pacific. As in 2002, a new GGII.4 variant detected in the spring and summer corresponded with high norovirus activity in the subsequent winter. Because outbreaks on cruise ships are likely to occur when new variants circulate, an active reporting system could function as an early warning system. Internationally accepted guidelines are needed for reporting, investigating, and controlling norovirus illness on cruise ships in Europe.

Host genetic resistance to symptomatic norovirus (GGII.4) infections in Denmark.

A total of 61 individuals involved in five norovirus outbreaks in Denmark were genotyped at nucleotides 428 and 571 of the FUT2 gene, determining secretor status, i.e., the presence of ABH antigens in secretions and on mucosa. A strong correlation (P = 0.003) was found between the secretor phenotype and symptomatic disease, extending previous knowledge and confirming that nonsense mutations in the FUT2 gene provide protection against symptomatic norovirus (GGII.4) infections.

Confirmation of electron microscopy results by direct testing of viruses adhered to grids using nucleic acid amplification techniques.

It is possible to visualize rapidly viral particles by electron microscopy (EM) in patient samples and in cell cultures, and characterize the particles on the basis of their size and morphology. In many instances, EM has contributed to the diagnosis of specific infectious agents. Four different types of viruses with different characteristics of particle size, capsid structure, the presence or absence of an envelope, genomic content and stability outside the host were screened and diagnosed by EM at the level of family/genus. The results were confirmed at the species level by elution of the sample material from the grids used for EM examination and nucleic acid amplification. This approach could be valuable in situations where the immediate diagnosis is unclear, or when new infectious agents appear.

An unusual enterovirus outbreak in Denmark: clinical characteristics and molecular epidemiology.

In 2000, a large enterovirus (EV) outbreak was seen in Denmark; the number of patients with a verified EV infection was 3-fold higher compared to previous y. Echovirus 30 (E30) was the dominant EV type and was detected in 31% of all 306 EV positive patients and in 61% of the 155 patients in whom typing was successful. The outbreak started in February and peaked in June, which is unusually early in a temperate climate and not registered before in Denmark. The age distribution of the patients also differed from previous y with a significantly higher proportion of older children and adults being affected. The patients had mainly symptoms consistent with aseptic meningitis. A phylogenetic analysis based upon a part of the VP1 structural gene of 21 E30 isolates showed that the Danish isolates belonged to the E30 genotype which has prevailed in Europe during the last few years. However, they constituted a separated cluster compared with 2 other outbreaks in other parts of Europe in 2000.

The porcine skin associated T-cell homing chemokine CCL27: molecular cloning and mRNA expression in piglets infected experimentally with Staphylococcus hyicus.

CCL27 (also named CTACK, ALP, ILC and ESkine) is a CC chemokine primarily expressed by keratinocytes of the skin. The cognate receptor of CCL27 named CCR10 (GPR-2), is also expressed in skin-derived cells, and in addition by a subset of peripheral blood T-cells and in a variety of other tissues. In this paper, we report the cloning of porcine CCL27 cDNA and investigation of CCL27 mRNA expression in Staphylococcus hyicus infected piglets. At the protein level, 77 and 74% homology was found to human and mouse CCL27 sequences, respectively. The results of the expression analyses show that CCL27 mRNA is upregulated in the skin of infected piglets and to a lesser extent in piglets recovered from disease and without clinical signs of infection, indicating a role for CCL27 both during inflammation and after recovery from an infection.