Showing papers by "Reimar Johne published in 2016"

Proposed reference sequences for hepatitis E virus subtypes

Abstract: The nomenclature of hepatitis E virus (HEV) subtypes is inconsistent and makes comparison of different studies problematic. We have provided a table of proposed complete genome reference sequences for each subtype. The criteria for subtype assignment vary between different genotypes and methodologies, and so a conservative pragmatic approach has been favoured. Updates to this table will be posted on the International Committee on Taxonomy of Viruses website (http://talk.ictvonline.org/r.ashx?C). The use of common reference sequences will facilitate communication between researchers and help clarify the epidemiology of this important human pathogen. This subtyping procedure might be adopted for other taxa of the genus Orthohepevirus.

A taxonomy update for the family Polyomaviridae.

Abstract: Many distinct polyomaviruses infecting a variety of vertebrate hosts have recently been discovered, and their complete genome sequence could often be determined. To accommodate this fast-growing diversity, the International Committee on Taxonomy of Viruses (ICTV) Polyomaviridae Study Group designed a host- and sequence-based rationale for an updated taxonomy of the family Polyomaviridae. Applying this resulted in numerous recommendations of taxonomical revisions, which were accepted by the Executive Committee of the ICTV in December 2015. New criteria for definition and creation of polyomavirus species were established that were based on the observed distance between large T antigen coding sequences. Four genera (Alpha-, Beta, Gamma- and Deltapolyomavirus) were delineated that together include 73 species. Species naming was made as systematic as possible - most species names now consist of the binomial name of the host species followed by polyomavirus and a number reflecting the order of discovery. It is hoped that this important update of the family taxonomy will serve as a stable basis for future taxonomical developments.

Thermal Stability of Hepatitis E Virus as Estimated by a Cell Culture Method

Abstract: Hepatitis E virus (HEV) is an increasingly recognized zoonotic pathogen. Transmission is suspected to occur from infected pigs or wild boars to humans through direct contact, environmental pathways, or contaminated food. However, the physical and chemical stability of HEV is largely unknown, because suitable cell culture methods for infectivity measurement are missing. Here, we developed a titration method using infection of the cell line A549/D3 with HEV genotype 3 strain 47832c and subsequent counting of focus-forming units by immunofluorescence, which allowed HEV infectivity measurements within a 4-log-dilution range. Long-term storage of HEV in cell culture medium at different temperatures indicated a phase of rapid virus inactivation, followed by a slower progression of virus inactivation. Infective HEV was detected up to 21 days at 37°C, up to 28 days at room temperature, and until the end of the experiment (56 days) with a 2.7-log decrease of infectious virus at 4°C. Heat treatment for 1 min resulted in moderate decreases of infectivity up to 60°C, 2- to 3.5-log decreases between 65°C and 75°C, and no remaining virus was detected at temperatures of ≥80°C. Heating for 70°C resulted in a 3.6-log decrease after 1.5 min and the absence of detectable virus (>3.9-log decrease) after 2 min. The data were used to calculate predictive heat inactivation models for HEV. The results may help estimate HEV stability in the environment or food. The established method may be used to study other aspects of HEV stability in the future. IMPORTANCE In this study, a cell culture method was developed which allows the measurement of hepatitis E virus (HEV) infectivity. Using this system, the stability of HEV at different time-temperature combinations was assessed, and a predictive model was established. The obtained data may help estimate HEV stability in the environment or food, thus enabling an assessment of the relative risks of HEV infection through distinct routes and by distinct types of food in the future.

Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

Abstract: Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown.

Generation of an Avian-Mammalian Rotavirus Reassortant by Using a Helper Virus-Dependent Reverse Genetics System

Abstract: The genetic diversity of rotavirus A (RVA) strains is facilitated in part by genetic reassortment. Although this process of genome segment exchange has been reported frequently among mammalian RVAs, it remained unknown if mammalian RVAs also could package genome segments from avian RVA strains. We generated a simian RVA strain SA11 reassortant containing the VP4 gene of chicken RVA strain 02V0002G3. To achieve this, we transfected BSR5/T7 cells with a T7 polymerase-driven VP4-encoding plasmid, infected the cells with a temperature-sensitive SA11 VP4 mutant, and selected the recombinant virus by increasing the temperature. The reassortant virus could be stably passaged and exhibited cytopathic effects in MA-104 cells, but it replicated less efficiently than both parental viruses. Our results show that avian and mammalian rotaviruses can exchange genome segments, resulting in replication-competent reassortants with new genomic and antigenic features. IMPORTANCE This study shows that rotaviruses of mammals can package genome segments from rotaviruses of birds. The genetic diversity of rotaviruses could be broadened by this process, which might be important for their antigenic variability. The reverse genetics system applied in the study could be useful for targeted generation and subsequent characterization of distinct rotavirus reassortant strains.

Norovirus outbreak in a restaurant: investigation of the path of infection by sequence analysis of food and human samples

Abstract: In September 2014, a small gastroenteritis outbreak occurred in a Greek restaurant. Primary investigations by official food surveillance revealed significant hygienic problems in the premises. Food samples and environmental samples were analyzed for the presence of bacterial and viral food pathogens. Norovirus genogroup I (GI) was detected in 2 environmental samples and in mixed salad. At the same time, stool samples from patients were analyzed and norovirus GI was detected. Further investigations revealed the presence of norovirus GI on some of the restaurant employees. Comparison of nucleic acid sequences revealed full sequence homology between norovirus RNA genotype 1.2 in food, environmental and stool samples, suggesting a common source of contamination and infection. Sequence analysis of food and environmental samples was facilitated by application of a system for total RNA amplification. Despite the fact that original source of contamination could be determined doubtlessly, observed weaknesses in the food production that caused this outbreak were discussed. The mixed salad could have been contaminated either by the lettuce contaminated at primary production or by one of the food-handlers. The investigation of the path of infection is necessary for the kind of legal consequences to be directed by authorities and may contribute to measures to eliminate possible sources of food contamination.