Identification of a novel astrovirus in domestic sheep in Hungary

The family Astroviridae consists of two genera, Avastrovirus and Mamastrovirus, whose members are associated with gastroenteritis in avian and mammalian hosts, respectively. We serendipitously identified a novel ovine astrovirus in a fecal specimen from a domestic sheep (Ovis aries) in Hungary by viral metagenomic analysis. Sequencing of the fragment indicated that it was an ORF1b/ORF2/3′UTR sequence, and it has been submitted to the GenBank database as ovine astrovirus type 2 (OAstV-2/Hungary/2009) with accession number JN592482. The unique sequence characteristics and the phylogenetic position of OAstV-2 suggest that genetically divergent lineages of astroviruses exist in sheep.     

Detection and genetic characterization of a novel pig astrovirus: relationship to other astroviruses

Emerging viruses represent a continuous threat to human health and to farmed animals, as evidenced on multiple occasions by outbreaks of influenza, henipavirus and SARS. Knowledge about the diversity of viromes present in reservoir species can lead to a better understanding of the origin of emerging pathogens. In this study, we extend the knowledge of astrovirus diversity in pigs by reporting the genetic characterization of an unknown astrovirus lineage. Phylogenetic analyses provided evidence that this porcine astrovirus lineage is unique and does not appear to share a recent common ancestor with any known mamastrovirus. The data reported in this study extend the number of porcine astrovirus lineages to a total of five, all of which most likely represent distinct species of different origins.     

Complete sequence of a novel duck astrovirus

A duck astrovirus isolate CPH (DAstV/CPH) detected from newly hatched ducklings was sequenced. The 7463-nt-long genome contained three open reading frames (ORFs) that were in three different frames, as seen in DAstV/C-NGB. Sequence comparison and phylogenetic analysis based on the full-length amino acid sequences of the three ORFs demonstrated that DAstV/CPH was highly divergent from previously known avastroviruses for which corresponding sequences are available. Genetic analysis of the complete ORF2 region revealed that mean amino acid genetic distances shared by DAstV/CPH with members of the three established avastrovirus species and other avastroviruses (except the chicken astrovirus GA2011 [CAstV/GA2011]-like viruses) were between 0.577 and 0.787, suggesting that DAstV/CPH may represent an additional avastrovirus species. The mean amino acid genetic distance between DAstV/CPH and the CAstV/GA2011-like viruses was 0.366, demonstrating the difficulty in determining the taxonomic relationship of DAstV/CPH to the CAstV/GA2011-like viruses. Interestingly, analysis based on a portion of the ORF1b region indicated that DAstV/CPH was more closely related to the common-teal-origin astrovirus MPK514 than to the CAstV/GA2011-like viruses. The findings are likely to provide new insights into the ecology and evolution of avastroviruses.     

Identification of a novel variant of human hepatitis E virus in Hungary.

First human case of hepatitis E infection detected in Hungary is reported. This hepatitis E virus (HEV), Hungary1, belongs to genotype 3 and had 95% and 90% nucleotide identity within the capsid region of the European swine and human (Greece2) strains, respectively. Hungary1 represents a potential novel human variant of HEV in genus Hepevirus.     

Enteric disease in dogs naturally infected by a novel canine astrovirus

Infection by a novel canine astrovirus was associated with gastroenteritis in two dogs. The virus displayed 70.3 to 73.9% amino acid identity to other canine astroviruses in the full-length capsid. Specific antibodies were detected in the convalescent-phase sera of the dogs, indicating seroconversion. Also, the virus appeared weakly related antigenically to the prototype canine astrovirus isolate ITA/2008/Bari.     

Identification of structural domains involved in astrovirus capsid biology

Coat proteins of non-enveloped, icosahedral viruses must perform a variety of functions during their life cycle such as assembly of the coat protein subunits into a closed shell, specific encapsidation of the viral nucleic acid, maturation of the capsid, interaction with host receptors, and disassembly to deliver the genetic information into the newly infected cell. A thorough understanding of the multiple capsid properties at the molecular level is required in order to identify potential targets for antiviral therapy and the prevention of viral disease. The system we have chosen for study is the astrovirus, a family of icosahedral, single-stranded RNA viruses that cause disease in mammals and birds. Very little is known about what regions of the coat protein contribute to the diverse capsid functions. This review will present novel structural predictions for the coat protein sequence of different astrovirus family members. Based on these predictions, we hypothesize that the assembly and RNA packaging functions of the astrovirus coat protein constitutes an individual domain distinct from the determinants required for receptor binding and internalization. Information derived from these structural predictions will serve as an important tool in designing experiments to understand astrovirus biology.     

Phylogenetic analysis of porcine astrovirus in domestic pigs and wild boars in South Korea

Porcine astrovirus (PAstV) belongs to genetically divergent lineages within the genus Mamastrovirus. In this study, 25/129 (19.4 %) domestic pig and 1/146 (0.7 %) wild boar fecal samples tested in South Korea were positive for PAstV. Positive samples were mainly from pigs under 6 weeks old. Bayesian inference (BI) tree analysis for RNA-dependent RNA polymerase (RdRp) and capsid (ORF2) gene sequences, including Mamastrovirus and Avastrovirus, revealed a relatively geographically divergent lineage. The PAstVs of Hungary and America belong to lineage PAstV 4; those of Japan belong to PAstV 1; and those of Canada belong to PAstV 1, 2, 3, and 5, but not to 4. This study revealed that the PAstVs of Korea belong predominantly to lineage PAstV 4 and secondarily to PAstV 2. It was also observed that PAstV infections are widespread in South Korea regardless of the disease state in domestic pigs and in wild boars as well.     

Contribution of clinical trials to gross domestic product in Hungary

Aim

To determine the contribution of clinical trials to the gross domestic product (GDP) in Hungary.

Methods

An anonymous survey of pharmaceutical companies and clinical research organizations (CROs) was conducted to estimate their clinical trial-related employment and revenues. Clinical trial documents at the National Institute of Pharmacy (NIP) were analyzed to estimate trial-related revenues at health care institutions and the value of investigational medical products (IMPs) based on avoided drug costs. Financial benefits were calculated as 2010 US $ purchasing power parity (PPP) values.

Results

Clinical trials increased the revenue of Hungarian health care providers by US $165.6 million. The value of IMPs was US $67.0 million. Clinical trial operation and management activities generated 900 jobs and US $166.9 million in revenue among CROs and pharmaceutical companies.

Conclusions

The contribution of clinical trials to the Hungarian GDP in 2010 amounted to 0.2%. Participation in international clinical trials may result in health, financial, and intangible benefits that contribute to the sustainability of health care systems, especially in countries with severe resource constraints. Although a conservative approach was employed to estimate the economic benefits of clinical trials, further research is necessary to improve the generalizability of our findings.Active participation in international clinical trials may provide health benefits to patients and financial and professional benefits to health care providers. In lower income economies, such as those in Central-Eastern Europe (CEE), the relative benefits of clinical trials are even greater than in the high income countries of Western Europe and North America. Consequently, the contribution of emerging markets to international clinical trials is growing substantially (1). This phenomenon is especially visible in CEE, where the number of clinical trials has increased significantly over the past 15 years and is expected to increase even further in the near future (2). In CEE, international clinical trials offer opportunities for site personnel to improve their professional networking and be remunerated on higher-than average income level. For health care institutions with substantial budget constraints, trial-related payments can represent an important source of liquid cash. A supportive attitude of hospital management toward clinical trial activities, in terms of providing better working environment or increased remuneration, may help to prevent the migration of qualified professional staff to higher income countries. In CEE countries, the health status of the population is worse than in higher income Western European countries (3) and the accessibility of new medicines is relatively limited (4). Therefore, through clinical trials, CEE patients can obtain access to standardized modern health care services, technologies, and investigational drugs without waiting lists or co-payments. However, investigational medical products (IMPs) may represent considerable health risks for patients.The societal gain associated with clinical trials is multifactorial. Clinical trials contribute to the evolution of evidence-based medicine. They systematically investigate side effects and health outcomes not only for IMPs but also for the control treatment arms. Therefore, safety information, even about marketed therapies, is captured and no public investment is necessary.The most tangible benefit may be the financial impact, including the contribution of trials to the revenues of health care providers and clinical research organizations (CROs). However, there are also indirect benefits, such as avoided health care expenses due to the free delivery of IMPs and services.Few scientific publications have addressed the financial benefits of clinical trials. These publications examined avoided drug costs and additional revenues primarily from the viewpoint of health care institutions (5-9). There is also one Polish study on the national economic impact of clinical trials, but the approach was not comprehensive enough to capture all direct and indirect financial benefits (10).Hungary currently has a favorable position for implementation of clinical trials (11). It has high-level professionalism at investigational centers, rapid regulatory and ethical endorsements of applications, complex but manageable contracting processes at clinical sites, sufficient contributions to patient recruitment, and high Good Clinical Practice (GCP) quality according to Food and Drug Administration (FDA) inspections (12). However, similarly to other CEE countries, the capacity for clinical trial participation in Hungary has not been maximized. The aim of this study was to determine the contribution of clinical trials to the national economy in Hungary. We estimated the clinical trial-related revenues of CROs, investigators, and health care institutions and the financial benefits of avoided drug costs due to IMPs as the percentage of the gross domestic product (GDP).     

Characterization of a novel porcine enterovirus in wild boars in Hungary

Porcine enteroviruses (PEVs) are members of the family Picornaviridae, genus Enterovirus. Until now, only three different PEV genotypes (PEV-9 and -10, and PEV-3H/PEV-14) have been detected in domestic pigs, and there is no information about the presence of PEVs in wild animals. Here, we identify and characterize the complete genomes of PEV originated from 5 of 10 (50%) of wild boar (Sus scrofa) piglets by RT-PCR and pyrosequencing. Wild boar/WBD/2011/HUN (JN807387) PEV showed only 67% amino acid identity in VP1 compared to the most closely related prototype PEV-3H/PEV-14. Wild boar enterovirus represents a novel PEV genotype, provisionally called PEV-15.     

Molecular characterization of a novel recombinant strain of human astrovirus associated with gastroenteritis in children

Summary.  We report a naturally occurring human astrovirus (HAstV) strain detected in two different geographic locations. We identified two isolates of this strain in a diarrhea outbreak at a child care center in Houston, Texas; and two isolates in diarrhea stool samples from two children in Mexico City. All four isolates were detected in stool samples by enzyme immunoassay (EIA). One of the Mexican isolates was typed by EIA and all four isolates were HAstV-5 by typing RT-PCR. The four isolates were >97% nucleotide-identical in two different genomic regions: ORF1a (246nt), and the 3′ end of the genome (471nt). One isolate from each geographic location was further sequenced in the transition region from ORF1b to ORF2 (1255nt) and this region of the two isolates showed ≥ 99% nt identity. Phylogenetic analyses of sequences of eight HAstV antigenic types and the novel strain in the transition region demonstrated the new strain being closely related to HAstV-3 in ORF1b, but closest to HAstV-5 in ORF2. These results and high sequence identity among all HAstV antigenic types in the transition region and RNA structural predictions supported a potential recombination site at the ORF1b/ORF2 junction. This is the first evidence that recombination occurs among human astroviruses. Received December 13, 2000 Accepted July 5, 2001     

Identification of novel Helicobacter species in pig stomachs by PCR and partial sequencing

Evidence of infection with Helicobacter species in pig stomach was investigated by the use of a PCR with Helicobacter genus-specific primers. Forty pig stomachs, each of four different ulcer lesion grades, 0, 1, 2, and 3 in the pars esophagea area, were collected from a slaughterhouse in Minnesota. Of 160 stomach samples examined, 102 (63.8%) were positive by the PCR assay. The 40 samples each of lesion grades 0, 1, 2, and 3 showed 22.5, 52.5, 85.0, and 95.0% PCR-positive results, respectively. There was a significant trend (P < or = 0.01) in the proportions of PCR-positive cases relative to severity of the lesion. About 80% of the 16S rRNA gene was amplified, and PCR-restriction fragment length polymorphism (RFLP) patterns were analyzed. Of 102 PCR-positive samples, the PCR-RFLP patterns resulted in four different types, 32 samples being classified into type MN 1, 16 samples into type MN 2, 43 samples into type MN 3, and 11 samples into type MN 4. When the sequences of each RFLP type were compared to those reported in databases by using BLAST software, types MN 1, MN 2, MN 3, and MN 4 showed homologies of 97.3, 95.4, 96.7, and 99.5% with the 16S ribosomal DNA of Helicobacter flexispira taxon 3, Helicobacter sp. strains MIT 94-022 and MZ 640285, and Helicobacter suis, respectively. None of the 102 samples positive for the Helicobacter genus were positive with a primer set specific for Helicobacter pylori. Attempts to culture the organisms from selected stomachs in vitro were unsuccessful.     

Identification of a novel cat allergen--cystatin

BACKGROUND: Cat allergen is an important cause of sensitization among children with asthma in Japan. Although there is good evidence that cats produce other allergens, only one major allergen, Fel d 1, has been studied in detail. AIMS: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using IgE antibodies to cat dander and selected clones were sequenced and expressed. RESULTS: One cDNA clone contained an open reading frame encoding a 98-amino acid residue protein. Sequence homology searches revealed a high degree of identity with bovine and human cystatin A, 79 and 75%, respectively. This cat cystatin clone contained the conserved cysteine protease motif and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE Ab to cat cystatin. This cysteine protease inhibitor motif was partially conserved in dog allergens, Can f 1 and Can f 2, which are lipocalins. Recombinant cystatin was produced in Escherichia coli cells and purified as an 11-kD protein, corresponding to the predicted MW of cystatin. The structure of cat cystatin was modeled on human cystatin B using the SWISS-MODEL. CONCLUSION: A newly identified allergen, cystatin, has been cloned from cat skin and is a member of the cysteine protease inhibitor family.     

Molecular characterization of a porcine astrovirus strain in China

Pigs are increasingly recognized to harbor a wide range of viruses that apparently establish long-term persistence in these animals. They serve as reservoirs for a number of human zoonotic diseases. In this study, a porcine astrovirus (PAstV) strain, designated as PAstV JWH-1, is identified from a diarrheal pig in China, and it is partially characterized genetically. Sequence analysis shows that the PAstV JWH-1 strain contains divergent nucleotide sequences in both the open reading frame (ORF)1b/ORF2 consensus and the 3′-UTR regions (s2m motif), which are usually highly conserved among members of the family Astroviridae. Phylogenetic analysis indicates that the JWH-1 strain clusters closely with newly identified strains PAstV 12-4 and 14-4 and forms a group of mamastroviruses with the proposed novel deer astrovirus. Further recombination analysis shows that two possible interspecies recombination events between porcine and deer astroviruses occurred in the genome of the JWH-1 strain. This study further confirms that multiple lineages are present among PAstVs, and each lineage likely represents an independent origin. Additionally, the possibility of interspecies transmission among PAstVs is also suggested.     

Detection of astrovirus gastroenteritis in children

A commercial enzyme immunoassay (EIA) for the detection of astrovirus antigen was used to detect the virus during a 12-month survey of enteric pathogens in children in outpatient (n = 238) and hospital (n = 176) settings. It was found to have a 100% sensitivity and 98.6% specificity. Nineteen astrovirus isolates were detected and confirmed by northern hybridization, cell culture, and RT-PCR. The virus was detected mainly amongst outpatients although a comparison of the detection rate with that in hospitalised children did not demonstrate a statistically significant difference (p = 0.1347). In contrast, there was a strong association between hospitalization and rotavirus infection (p = 0.0371), and a strong association between infection detected in outpatients and adenovirus infection (p = 0.0193). Strains of astrovirus were sequenced, genotyped and shown to be: type 1 (n = 11), type 3 (n = 1), and type 4 (n = 7). Maximum genetic variation in type 1 isolates was 8.6% and type 4 was 7.8%. Changes did not result in amino acid substitutions.     

Pathogenesis of astrovirus infection

Astroviruses are one of the leading causes of diarrhea worldwide. In spite of its impact on human health, little is known about astrovirus pathogenesis. One reason for this may be the lack of a suitable small animal model for infection. In recent years, there has been increasing information on the mechanism of astrovirus-induced disease in mammals (including humans) and birds. This review summarizes our current state of knowledge on astrovirus pathogenesis.