Evidence of Circulation and Phylogenetic Analysis of Hepatitis E Virus (HEV) in Wild Boar in South-East Italy

La Bella, Gianfranco; Basanisi, Maria Grazia; Nobili, Gaia; Coppola, Rosa; Damato, Annita Maria; Donatiello, Adelia; Occhiochiuso, Gilda; Romano, Antonella Cristina; Toce, Mariateresa; Palazzo, Lucia; Pellegrini, Francesco; Fanelli, Angela; Di Martino, Barbara; Suffredini, Elisabetta; Lanave, Gianvito; Martella, Vito; La Salandra, Giovanna

doi:10.3390/v15102021

Cited by 3 publications

(1 citation statement)

References 68 publications

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“…In order to evaluate the sensitivity of the assay, the ddPCR protocol was applied to a selection of naturally HEV-contaminated wild boar muscle samples derived from hunting activities and intended for human consumption from the Apulia and Basilicata regions in the year 2022. These samples were previously analyzed using an RT-qPCR method, as previously described [ 3 , 32 ]. In detail, 36 samples were analyzed, of which 18 resulted positive and 18 resulted negative using the RT-qPCR.…”

Section: Methodsmentioning

confidence: 99%

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food

La Bella,

Basanisi,

Nobili

et al. 2024

Viruses

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Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.

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Section: Methodsmentioning

confidence: 99%

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food

La Bella,

Basanisi,

Nobili

et al. 2024

Viruses

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Longitudinal serological and virological survey of hepatitis E virus in wild boar (Sus scrofa majori, Maremman wild boar) and fallow deer (Dama dama) populations in a protected area of Central Italy

Luca,

Mariagiovanna,

Paola

et al. 2024

Front. Vet. Sci.

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Hepatitis E virus (HEV) is recognized as an emerging zoonosis. Pigs and wild boars are considered the main reservoirs of zoonotic HEV-3 and HEV-4 genotypes. In Europe, autochthonous human cases of hepatitis E, mainly associated with HEV-3 and consumption of raw or undercooked pig and wild boar liver/meat, have increased over the last decades. From 2016 to 2024, during several hunting seasons, we conducted a molecular and serological longitudinal survey on the circulation of HEV in Maremman wild boar (Italian subspecies/ecotype, Sus scrofa majori) and fallow deer (Dama dama) populations in a protected area in Central Italy. During the study period, 346 livers (256 from wild boar, 90 from fallow deer), 161 serum (127 from wild boar, 34 from fallow deer), and 23 meat juice (11 from wild boar, 12 from fallow deer) samples were collected. Serum and meat juice samples were tested using a commercial ELISA test for the detection of total anti-HEV antibodies. An estimated serological prevalence of 28.3% (39/138) in wild boar and 21.7% (10/46) in fallow deer was found. The 346 liver samples were tested using a HEV Real-Time RT-PCR for the detection of HEV-RNA. Thirty-one wild boar (12%) and four fallow deer (4.4%) livers were found positive. Phylogenetic analysis of 11 partial ORF2 sequences from wild boar confirmed the HEV3 heterogeneity in this species, revealing different strains (3f, 3c) circulating over the years. The detected subtypes are among the most commonly detected in Italy and our strains showed a high correlation with human and wild boar Italian strains. Although the studied area is a fenced natural reserve, the presence of different strains over time suggests the probable virus introduction from the external. Our results confirm fallow deer susceptibility to the infection, and that wild boar could be considered the main wild HEV reservoir. This is also the first study demonstrating the infection in the so-called Italian subspecies/ecotype Maremman wild boar. Moreover, our results corroborate that the consumption of undercooked or raw liver from both wild boar and fallow deer, or the direct contact with these animals, could represent a zoonotic risk.

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Design of primer and linear probe sets for swine and wild boar HEV detection by PCR

Lymanska

2024

Veterinary Medicine: Inter-Departmental Subject Scientific Collection

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Hepatitis E virus (HEV) infects humans and several mammals and it has 8 genotypes (HEV1 HEV8). HEV1 4 is the causative agent of Hepatitis E in humans. HEV5 6 was detected only in wild boar in Japan, HEV7 8 was detected in camels. HEV3 4 is characterized by zoonotic potential. Swines and wild boars are main natural reservoirs for this virus. Besides, HEV 3 was detected in deers, dolphins, rabbits, cattle, goats that is additional risk for virus interspecies transmission from domestic animals to humans. Availability of mismatched nucleotides in the complexes of primer/probe with viral targets was applied for estimation of primer sets. Based on determined conserved fragments two sets with LNA containing primer and linear LNA containing probe without mismatched nucleotides in the complexes of primer/probe with single stranded amplicon for real time reverse transcriptase PCR detection of swine and wild boar HEV 3 and HEV 4 isolates were designed. Primer sets may be used for HEV detection by standard PCR

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Evidence of Circulation and Phylogenetic Analysis of Hepatitis E Virus (HEV) in Wild Boar in South-East Italy

Cited by 3 publications

References 68 publications

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food

Longitudinal serological and virological survey of hepatitis E virus in wild boar (Sus scrofa majori, Maremman wild boar) and fallow deer (Dama dama) populations in a protected area of Central Italy

Design of primer and linear probe sets for swine and wild boar HEV detection by PCR

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