Thermal stability of hepatitis E virus assessed by a molecular biological approach

Background: Hepatitis E virus (HEV) is a pathogen of emerging concern in industrialized countries. The consumption of wild boar meat has been identified as one risk factor for autochthonous HEV infections. Only limited information is available about thermal stability of HEV, mainly due to the lack of rapid and efficient cell culture systems for measurement of HEV infectivity. Methods: A molecular biological method was implemented in order to distinguish disassembled from intact viral particles using RNase treatment followed by quantitative real-time RT-PCR. The method was applied to a wild boar liver suspension containing HEV genotype 3. Results: Time-course analyses indicated that the decline of protected RNA could be described by a biphasic model with an initial decrease followed by a stationary phase. The stationary phase was reached after 1 hour at 4°C, 3 days at 22°C and 7 days at 37°C with log reductions of 0.34, 0.45 and 1.24, respectively. Protected RNA was detectable until the end of the experiments at day 50 or 70. Heat exposure for 1 minute resulted in a log reduction of 0.48 at 70°C and increased with higher temperatures to 3.67 at 95°C. Although HEV infectivity titration by inoculation of the liver suspension onto three cell lines did not succeed, the results of the RNase-based method are in accordance with published cell culture-based data. Conclusions: Measurement of intact viral particles using the RNase-based method may provide data on the stability of RNA viruses when cell culture-based infectivity titrations are not efficient or not available. The method enables processing of large sample numbers and may be suitable to estimate stability of HEV in different types of food

Detection of hepatitis E virus in wild boars of rural and urban regions in Germany and whole genome characterization of an endemic strain

<p>Abstract</p> <p>Background</p> <p>Hepatitis E is an increasingly diagnosed human disease in Central Europe. Besides domestic pigs, in which hepatitis E virus (HEV) infection is highly prevalent, wild boars have been identified as a possible source of human infection. In order to assess the distribution of HEV in the wild boar population of Germany, we tested liver samples originating from different geographical regions for the presence of the HEV genome and compared the detected sequences to animal and human HEV strains.</p> <p>Results</p> <p>A total of 148 wild boar liver samples were tested using real-time RT-PCR resulting in an average HEV detection rate of 14.9% (95% CI 9.6–21.6). HEV was detected in all age classes and all geographical regions. However, the prevalence of HEV infection was significantly higher in rural as compared to urban regions (p < 0.001). Sequencing of the PCR products indicated a high degree of heterogenicity of the detected viruses within genotype 3 and a grouping according to their geographical origin. The whole genome sequence of an HEV isolate (wbGER27) detected in many wild boars in the federal state of Brandenburg was determined. It belongs to genotype 3i and shows 97.9% nucleotide sequence identity to a partial sequence derived from a human hepatitis E patient from Germany.</p> <p>Conclusion</p> <p>The results indicate that wild boars have to be considered as a reservoir for HEV in Germany and that a risk of HEV transmission to humans is present in rural as well as urban regions.</p

High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (OR3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (OR3) upstream of the prophage repressor gene. The OR3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages

Hepatitis E virus antibody prevalence in hunters from a district in Central Germany, 2013: a cross-sectional study providing evidence for the benefit of protective gloves during disembowelling of wild boars

Background: In Germany, 17 % of the general human population have antibodies to hepatitis E virus (HEV) (recomLine HEV-IgG/IgM immunoassay [Mikrogen GmbH]). Wild boars represent an animal reservoir for HEV genotype 3, which is the common genotype in Germany. We estimated the seroprevalence among hunters with contact to wild boars to identify factors that may be associated with past or present HEV infection. Methods: In 2013, the local veterinarian authority in a district in Central Germany attended meetings of hunters who provided blood specimens and completed a questionnaire collecting information on age, sex, hunting-related activities and consumption of wild boar meat. Specimens of wild boars were taken during drive hunts in this district during the season 2012/2013. All specimens were tested for HEV RNA and anti-HEV IgM and IgG antibodies. Log-binomial regression was used to estimate prevalence ratios (PR) for the hunters. Results: Of 126 hunters (median age 55; 94 % male) 21 % tested positive for anti-HEV IgG antibodies (95 % confidence interval [CI] 13–28 %) (recomWell HEV IgG assay [Mikrogen GmbH]). Anti-HEV prevalence was highest in the age group of the 70–79-year-olds (67 %; 95 % CI 39–95 %). Wild boars showed an average anti-HEV prevalence of 41 %. HEV RNA was detected in 4/22 (18 %) liver specimens and in 1/22 (4.5 %) muscle specimens. Most wild boars were tested positive for HEV RNA (3/10; 30 %) and HEV-specific antibodies (7/15; 47 %) in the southwestern part of the district. Hunters preferring this hunting ground had a lower anti-HEV prevalence when gloves were frequently used during disembowelling of wild boars compared to hunters using gloves never or infrequently (age-adjusted PR 0.12; 95 % CI 0.02–0.86). Conclusions: Hunters may benefit from wearing gloves when in contact with blood or body fluids of HEV animal reservoirs. Anti-HEV prevalence among the hunters of this study did not significantly differ from that of the general population suggesting that other factors play a major role in the epidemiology of HEV in Germany

Isolation of Subtype 3c, 3e and 3f-Like Hepatitis E Virus Strains Stably Replicating to High Viral Loads in an Optimized Cell Culture System

The hepatitis E virus (HEV) is transmitted via the faecal–oral route in developing countries (genotypes 1 and 2) or through contaminated food and blood products worldwide (genotypes 3 and 4). In Europe, HEV subtypes 3c, 3e and 3f are predominant. HEV is the leading cause of acute hepatitis globally and immunocompromised patients are particularly at risk. Because of a lack of cell culture systems efficiently propagating wild-type viruses, research on HEV is mostly based on cell culture-adapted isolates carrying uncommon insertions in the hypervariable region (HVR). While optimizing the cell culture system using the cell culture-adapted HEV strain 47832c, we isolated three wild-type strains derived from clinical specimens representing the predominant spectrum of HEV in Europe. The novel isolates 14-16753 (3c), 14-22707 (3e) and 15-22016 (3f-like) replicate to high viral loads of 108, 109 and 106.5 HEV RNA copies/mL at 14 days post-inoculation, respectively. In addition, they could be kept as persistently infected cell cultures with constant high viral loads (~109 copies/mL) for more than a year. In contrast to the latest isolates 47832c, LBPR-0379 and Kernow-C1, the new isolates do not carry genome insertions in the HVR. Optimization of HEV cell culture identified amphotericin B, distinct salts and fetal calf serum (FCS) as important medium supplements. Overconfluent cell layers increased infectivity and virus production. PLC/PRF/5, HuH-7-Lunet BLR, A549 and HepG2/C3A supported replication with different efficiencies. The novel strains and optimized cell culture system may be useful for studies on the HEV life cycle, inactivation, specific drug and vaccine development

Feeding of the probiotic bacterium Enterococcus faecium NCIMB 10415 differentially affects shedding of enteric viruses in pigs

Effects of probiotic bacteria on viral infections have been described previously. Here, two groups of sows and their piglets were fed with or without feed supplementation of the probiotic bacterium Enterococcus faecium NCIMB 10415. Shedding of enteric viruses naturally occurring in these pigs was analyzed by quantitative real-time RT-PCR. No differences between the groups were recorded for hepatitis E virus, encephalomyocarditis virus and norovirus. In contrast, astrovirus was exclusively detected in the non-supplemented control group. Rotavirus was shedded later and with lower amounts in the probiotic piglet group (p < 0.05); rotavirus-shedding piglets gained less weight than non-infected animals (p < 0.05). Serum titres of anti-rotavirus IgA and IgG antibodies were higher in piglets from the control group, whereas no difference was detected between sow groups. Phenotype analysis of immune cell antigens revealed significant differences of the CD4 and CD8β (p < 0.05) as well as CD8α and CD25 (p < 0.1) T cell populations of the probiotic supplemented group compared to the non-supplemented control group. In addition, differences were evident for CD21/MHCII-positive (p < 0.05) and IgM- positive (p < 0.1) B cell populations. The results indicate that probiotic bacteria could have effects on virus shedding in naturally infected pigs, which depend on the virus type. These effects seem to be caused by immunological changes; however, the distinct mechanism of action remains to be elucidated

Novel Hepatitis E Virus Genotype in Norway Rats, Germany

Human hepatitis E virus infections may be caused by zoonotic transmission of virus genotypes 3 and 4. To determine whether rodents are a reservoir, we analyzed the complete nucleotide sequence of a hepatitis E–like virus from 2 Norway rats in Germany. The sequence suggests a separate genotype for this hepatotropic virus

Aspects of high hydrostatic pressure food processing: Perspectives on technology and food safety

The last two decades saw a steady increase of high hydrostatic pressure (HHP) used for treatment of foods. Although the science of biomaterials exposed to high pressure started more than a century ago, there still seem to be a number of unanswered questions regarding safety of foods processed using HHP. This review gives an overview on historical development and fundamental aspects of HHP, as well as on potential risks associated with HHP food applications based on available literature. Beside the combination of pressure and temperature, as major factors impacting inactivation of vegetative bacterial cells, bacterial endospores, viruses, and parasites, factors, such as food matrix, water content, presence of dissolved substances, and pH value, also have significant influence on their inactivation by pressure. As a result, pressure treatment of foods should be considered for specific food groups and in accordance with their specific chemical and physical properties. The pressure necessary for inactivation of viruses is in many instances slightly lower than that for vegetative bacterial cells; however, data for food relevant human virus types are missing due to the lack of methods for determining their infectivity. Parasites can be inactivated by comparatively lower pressure than vegetative bacterial cells. The degrees to which chemical reactions progress under pressure treatments are different to those of conventional thermal processes, for example, HHP leads to lower amounts of acrylamide and furan. Additionally, the formation of new unknown or unexpected substances has not yet been observed. To date, no safety-relevant chemical changes have been described for foods treated by HHP. Based on existing sensitization to non-HHP-treated food, the allergenic potential of HHP-treated food is more likely to be equivalent to untreated food. Initial findings on changes in packaging materials under HHP have not yet been adequately supported by scientific data

ICTV virus taxonomy profile : Sedoreoviridae 2022

Sedoreoviridae is a large family of icosahedral viruses that are usually regarded as non- enveloped with segmented (10–12 linear segments) dsRNA genomes of 18–26 kbp. Sedoreovirids have a broad host range, infecting mammals, birds, crustaceans, arthropods, algae and plants. Some of them have important pathogenic potential for humans (e.g. rotavirus A), livestock (e.g. bluetongue virus) and plants (e.g. rice dwarf virus).Instituto de BiotecnologíaFil: Matthijnssens, Jelle. University of Leuven; BélgicaFil: Attoui, Houssam. National Institute for Agricultural Research (INRA); FranciaFil: Bányai, Krisztián. Veterinary Medical Research Institute; HungríaFil: Brussaard, Corina P. D. NIOZ Royal Netherlands Institute for Sea Research; Países BajosFil: Brussaard, Corina P. D. University of Utrecht; Países BajosFil: Danthi, Pranav. Indiana University; Estados UnidosFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Dermody, Terence S. University of Pittsburgh. School of Medicine; Estados UnidosFil: Duncan, Roy. Dalhousie University; CanadáFil: Fāng, Qín. Wuhan Institute of Virology; ChinaFil: Johne, Reimar. German Federal Institute for Risk Assessment; AlemaniaFil: Mertens, Peter P. C. University of Nottingham; Reino UnidoFil: Jaafar, Fauziah Mohd. Ecole Nationale Vétérinaire d’Alfort; FranciaFil: Patton, John T. Indiana University; Estados UnidosFil: Sasaya, Takahide. National Agriculture and Food Research Organization; JapónFil: Suzuki, Nobuhiro. Okayama University. JapónFil: Wei, Taiyun. Fujian Agriculture and Forestry University; Chin