Interlaboratory Validation of a Detection Method for Hepatitis E Virus RNA in Pig Liver

Trojnar, E.; Contzen, Matthias; Moor, Dominik; Carl, Anja; Burkhardt, Sabine; Kilwinski, Jochen; Berghof-Jäger, Kornelia; Mormann, Sascha; Schotte, Ulrich; Kontek, Anne; Althof, Nadine; Mäde, Dietrich; Johne, Reimar

doi:10.3390/microorganisms8101460

Cited by 4 publications

(2 citation statements)

References 37 publications

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“…HEV RNA was extracted using the MagMAX TM CORE Nucleic Acid Purification Kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer's instructions. RNA was detected by RT-qPCR with 50 cycles according to Jothikumar et al (2006) [31], using bacteriophage MS2 as an internal process control [32].…”

Section: Rna Extraction and Rt-qpcrmentioning

confidence: 99%

Detection of Hepatitis E Virus Infections in Wild Boars in Southwest Germany Using a Stepwise Laboratory Diagnostic Approach

et al. 2022

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Hepatitis E virus (HEV) is the main cause of enterically transmitted hepatitis in humans worldwide. Among HEV, genotypes 3 and 4 are considered zoonotic agents associated with domestic pigs and wild boars, showing an increasing trend in Europe. The aim of this study is to contribute data on the prevalence of HEV in wild boars in Southwest Germany and to present a time and cost-effective two-step laboratory diagnostic approach for serological monitoring of blood samples. This method uses enzyme-linked immunosorbent assay (ELISA), followed by testing for HEV RNA by reverse transcription real-time PCR (RT-qPCR). A total of 2295 blood samples were collected in 234 municipalities in 12 counties in the period from 2016 to 2020. There was an overall seroprevalence of 10.8%, ranging from 3.6% to 17.5% per county and 7.5% to 14% per year. Retesting of these blood samples for HEV RNA revealed 15.7% viremic wild boars originating from 30 municipalities in 11 counties. Viremic wild boars were found in seven regional clusters, including 84% of the animals that tested positive for HEV. Seropositive animals <1 year of age were significantly more likely to be viremic than those >1 year. Further characterization of HEV RNA resulted in the identification of genotype 3. Altogether, serological monitoring of the blood samples, complemented by successive and targeted investigations into the presence of HEV RNA based on blood samples, provide reliable information on the seroprevalence and virus load in wild boars, which proved to be a relevant and persistent sylvatic reservoir for HEV.

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Section: Rna Extraction and Rt-qpcrmentioning

confidence: 99%

Detection of Hepatitis E Virus Infections in Wild Boars in Southwest Germany Using a Stepwise Laboratory Diagnostic Approach

et al. 2022

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“…RNA was extracted from liver samples as described [18] using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA from serum and cell culture supernatants was extracted using the NucliSens ® EasyMag ® system (Biomérieux, Nürtingen, Germany).…”

Section: Detection and Quantification Of The Hev Genomementioning

confidence: 99%

Cell Culture Isolation and Whole Genome Characterization of Hepatitis E Virus Strains from Wild Boars in Germany

et al. 2021

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Infection with hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. The HEV genotype 3 can be zoonotically transmitted from animals to humans, with wild boars representing an important reservoir species. Cell culture isolation of HEV is generally difficult and mainly described for human isolates so far. Here, five sera and five liver samples from HEV-RNA-positive wild boar samples were inoculated onto PLC/PRF/5 cells, incubated for 3 months and thereafter passaged for additional 6 weeks. As demonstrated by RT-qPCR, immunofluorescence and immune electron microscopy, virus was successfully isolated from two liver samples, which originally contained high HEV genome copy numbers. Both isolates showed slower growth than the culture-adapted HEV strain 47832c. In contrast to this strain, the isolated strains had no insertions in their hypervariable genome region. Next generation sequencing using an HEV sequence-enriched library enabled full genome sequencing. Strain Wb108/17 belongs to subtype 3f and strain Wb257/17 to a tentative novel subtype recently described in Italian wild boars. The results indicate that HEV can be successfully isolated in cell culture from wild boar samples containing high HEV genome copy numbers. The isolates may be used further to study the zoonotic potential of wild boar-derived HEV subtypes.

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Evaluation of an Automated Ultrafiltration System for Concentrating a Range of Viruses from Saline Waters

Singh,

Aw,

Rose

2024

Food Environ Virol

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Pathogenic viruses in environmental water are usually present in levels too low for direct detection and thus, a concentration step is often required to increase the analytical sensitivity. The objective of this study was to evaluate an automated filtration device, the Innovaprep Concentrating Pipette Select (CP Select) for the rapid concentration of viruses in saline water samples, while considering duration of process and ease of use. Four bacteriophages (MS2, P22, Phi6, and PhiX174) and three animal viruses (adenovirus, coronavirus OC43, and canine distemper virus) were seeded in artificial seawater, aquarium water, and bay water samples, and processed using the CP Select. The recovery efficiencies of viruses were determined either using a plaque assay or droplet digital PCR (ddPCR). Using plaque assays, the average recovery efficiencies for bacteriophages ranged from 4.84 ± 3.8% to 82.73 ± 27.3%, with highest recovery for P22 phage. The average recovery efficiencies for the CP Select were 39.31 ± 26.6% for adenovirus, 19.04 ± 11.6% for coronavirus OC43, and 19.84 ± 13.6% for canine distemper virus, as determined by ddPCR. Overall, viral genome composition, not the size of the virus, affected the recovery efficiencies for the CP Select. The small sample volume size used for the ultrafilter pipette of the system hinders the use of this method as a primary concentration step for viruses in marine waters. However, the ease of use and rapid processing time of the CP Select are especially beneficial when rapid detection of viruses in highly contaminated water, such as wastewater or sewage-polluted surface water, is needed. Graphical abstract

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Interlaboratory Validation of a Detection Method for Hepatitis E Virus RNA in Pig Liver

Cited by 4 publications

References 37 publications

Detection of Hepatitis E Virus Infections in Wild Boars in Southwest Germany Using a Stepwise Laboratory Diagnostic Approach

Detection of Hepatitis E Virus Infections in Wild Boars in Southwest Germany Using a Stepwise Laboratory Diagnostic Approach

Cell Culture Isolation and Whole Genome Characterization of Hepatitis E Virus Strains from Wild Boars in Germany

Evaluation of an Automated Ultrafiltration System for Concentrating a Range of Viruses from Saline Waters

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