Showing papers by "Reimar Johne published in 2014"

An ORF1-rearranged hepatitis E virus derived from a chronically infected patient efficiently replicates in cell culture

Abstract: Hepatitis E is an increasingly reported disease in industrialized countries. Studies on the replication cycle of hepatitis E virus (HEV) are hampered due to the lack of efficient and robust cell culture systems for this virus. We describe the successful isolation of HEV derived from a chronically infected kidney transplant patient held under immunosuppressive therapy. Inoculation of serum sample 47832 onto the human lung carcinoma cell line A549 resulted in the replication of the virus as shown by RT-qPCR. This novel human-derived HEV strain is closely related to a wild boar-derived genotype 3 strain, which did not replicate in A549 cells. It carries a 186 nucleotide insertion in the hypervariable ORF1-region, derived from two parts of its ORF1. By passaging of the infected cells, a cell line continuously producing HEV particles was generated as demonstrated by RT-qPCR, immuno-electron microscopy, density gradient centrifugation and immunohistochemistry. Replication of the produced virus was demonstrated after its inoculation onto fresh A549 cells and two consecutive passages, whereas heating at 65 °C for 2 min abolished its infectivity. Several point mutations scattered along the whole genome were present in the HEV strain from the second passage; however, the ORF1 insertion was still present. Previously, cell culture isolation of two other HEV strains carrying insertions in their hypervariable regions, but originating from human ribosomal protein genes, has been described. The findings may indicate that cell culture adaptation of is mostly dependent on the length and position of the insertion, rather than from the sequence itself.

Metagenomic identification of novel enteric viruses in urban wild rats and genome characterization of a group A rotavirus.

Abstract: Rats are known as reservoirs and vectors for several zoonotic pathogens. However, information on the viruses shed by urban wild rats that could pose a zoonotic risk to human health is scare. Here, intestinal contents from 20 wild Norway rats (Rattus norvegicus) collected in the city of Berlin, Germany, were subjected to metagenomic analysis of viral nucleic acids. The determined faecal viromes of rats consisted of a variety of known and unknown viruses, and were highly variable among the individuals. Members of the families Parvoviridae and Picobirnaviridae represented the most abundant species. Novel picornaviruses, bocaviruses, sapoviruses and stool-associated circular ssDNA viruses were identified, which showed only low sequence identity to known representatives of the corresponding taxa. In addition, noroviruses and rotaviruses were detected as potential zoonotic gastroenteritis viruses. However, partial-genome sequence analyses indicated that the norovirus was closely related to the recently identified rat norovirus and the rotavirus B was closely related to the rat rotavirus strain IDIR; both viruses clustered separately from respective human virus strains in phylogenetic trees. In contrast, the rotavirus A sequences showed high identity to human and animal strains. Analysis of the nearly complete genome of this virus revealed the known genotypes G3, P[3] and N2 for three of the genome segments, whereas the remaining eight genome segments represented the novel genotypes I20–R11–C11–M10–A22–T14–E18–H13. Our results indicated a high heterogeneity of enteric viruses present in urban wild rats; their ability to be transmitted to humans remains to be assessed in the future.

Seroprevalence of hepatitis E virus (HEV) in humans living in high pig density areas of Germany

Abstract: An increase in acute autochthonous hepatitis E virus (HEV) infections has been recorded in Germany. These are suspected to be zoonotically transmitted from wild boar, deer and domestic pig. The latter may represent a major reservoir for HEV. In this study, 537 sera from humans living in Westphalia and Lower Saxony, representing areas of high pig density in Germany, were tested for the presence of HEV-specific antibodies. Among them were 302 individuals with occupational, direct contact to pigs and 235 individuals without direct contact to pigs. Two commercial tests and one in-house assay were applied for the detection of HEV-specific immunoglobulin G (IgG) antibodies. Sera were also tested in an assay that detects all classes of HEV-specific antibodies. Depending on the test used, the seroprevalence ranged from 4.1 to 27.9 %. Exposition to pigs was found to be associated with a significantly higher seroprevalence in subjects with contact to pigs (13.2-32.8 %) compared with that in non-exposed humans (7.7-21.7 %). In particular, individuals younger than 40 years with occupational exposure exhibited a markedly higher HEV seroprevalence compared with non-exposed individuals of that age group. In general, HEV seroprevalence increased with age resulting in a similar prevalence level in the age group of ≥ 50 years for exposed and non-exposed individuals. Analysis of all sera by a commercial anti-HEV IgM ELISA revealed 35 positive and 25 borderline samples. However, only one positive serum could be confirmed by an IgM line assay. Selected samples from IgM and/or IgG as well as total HEV antibody-positive individuals were also tested for the presence of HEV RNA. In one of the 78 samples, the only IgM ELISA positive and IgM line assay confirmed sample, RNA of HEV genotype 3 was detected. This sequence has high similarity to HEV sequences obtained from wild boars and domestic pigs from Germany and The Netherlands. This study demonstrates that in addition to the consumption of raw or undercooked meat, direct contact to pigs has to be considered as an additional risk factor for HEV infection.

The general composition of the faecal virome of pigs depends on age, but not on feeding with a probiotic bacterium.

Abstract: Background The pig faecal virome, which comprises the community of viruses present in pig faeces, is complex and consists of pig viruses, bacteriophages, transiently passaged plant viruses and other minor virus species Only little is known about factors influencing its general composition Here, the effect of the probiotic bacterium Enterococcus faecium (E faecium) NCIMB 10415 on the pig faecal virome composition was analysed in a pig feeding trial with sows and their piglets, which received either the probiotic bacterium or not Results From 8 pooled faecal samples derived from the feeding trial, DNA and RNA virus particles were prepared and subjected to process-controlled Next Generation Sequencing resulting in 390,650 sequence reads In average, 14% of the reads showed significant sequence identities to known viruses The percentage of detected mammalian virus sequences was highest (55–77%) in the samples of the youngest piglets and lowest (8–10%) in the samples of the sows In contrast, the percentage of bacteriophage sequences increased from 22–44% in the youngest piglets to approximately 90% in the sows The dominating mammalian viruses differed remarkably among 12 day-old piglets (kobuvirus), 54 day-old piglets (boca-, dependo- and pig stool-associated small circular DNA virus [PigSCV]) and the sows (PigSCV, circovirus and “circovirus-like” viruses CB-A and RW-A) In addition, the Shannon index, which reflects the diversity of sequences present in a sample, was generally higher for the sows as compared to the piglets No consistent differences in the virome composition could be identified between the viromes of the probiotic bacterium-treated group and the control group Conclusion The analysis indicates that the pig faecal virome shows a high variability and that its general composition is mainly dependent on the age of the pigs Changes caused by feeding with the probiotic bacterium E faecium could not be demonstrated using the applied metagenomics method