Project

Zoonotic viruses (HEV, MRV, and TTSuV) in wild fauna of Northern Italy

Goal: This project evaluates the occurrence of Hepatitis E virus (HEV), Mammalian orthoreovirus (MRV), and Torque teno sus virus (TTSuV) in wild animals in Northern Italy to better estimate the risk of transmission to farmed animals and possibly to humans.

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Sara Arnaboldi
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SUMMARY Hepatitis E virus (HEV) is an emerging virus with an increasing diffusion across Europe. Because of the apparent lack of species barriers, HEV was characterized as a zoonotic agent, with swine as main reservoir, but it has been also found in wild animals. This study aimed to assess the HEV prevalence in wild boars, hares and swine in an area of Northern Italy with high anthropic and farm density. Samples were represented by liver (wild boars and hares) and faeces (swine). Viral identification was carried out by RT Real-time PCR. HEV strains were genotyped, and phylogenetic analysis was performed. A prevalence of 1% was found in swine faeces. In wild fauna, HEV was found only in wild boars (prevalence 12.02%, subtype HEV-3a), that may play a role as reservoir. Given the high pig farm density in the investigated area, and the expansion of the wild boar population, continuous monitoring of the strains circulating in wildlife and swine is crucial. INTRODUZIONE Il virus dell'epatite E (HEV) è un virus emergente segnalato in tutta Europa. La sua variabilità genetica, la costante evoluzione e la presenza di numerosi ceppi sia nell'uomo che negli animali, contribuiscono alla sua diffusione e ad una potenziale espansione dei possibili ospiti. I genotipi 3 e 4 (HEV-3 e HEV-4) sono stati frequentemente associati allo sviluppo cronico della malattia e possono infettare sia l'uomo che gli animali (in particolare suini). Le principali vie di trasmissione sono quella alimentare (ingestione di carne di maiale o di selvaggina, cruda o poco cotta) e quella diretta (attraverso il contatto con animali infetti). In Italia, HEV-3 è stato identificato negli alimenti a base di carne di suino e di cinghiale (2,4) rendendo questi prodotti potenzialmente ad alto rischio di trasmissione di HEV. Inoltre, il possibile aumento della circolazione virale può essere influenzato dalla continua espansione dell'area di insediamento dei cinghiali, dall'elevata diffusione delle lepri e dall’alta densità di allevamenti di suini nella Pianura Padana. Lo scopo di questo studio è quindi indagare la presenza di HEV nei cinghiali, nelle lepri e nei suini in Pianura Padana, per chiarire la distribuzione di HEV ed il ruolo di questi animali nella sua circolazione. MATERIALI E METODI L'area studiata è la Pianura Padana, un’area ad alta diffusione di animali selvatici (circa 13-50 lepri/ha e 12 cinghiali/100 ha) e di allevamenti di suini. Il campionamento è avvenuto tra il 2018 e il 2022 e comprende lepri (Lepus europaeus), cinghiali (Sus scrofa) e scrofe (Sus scrofa domesticus). Dalle lepri e dai cinghiali sono stati prelevati campioni di fegato durante rispettivamente l’eviscerazione e le ispezioni post-mortem nei centri di lavorazione delle carni di selvaggina; per le scrofe, sono stati campionati dei pool di feci da due allevamenti in cui era stata effettuata una preliminare indagine sierologica per HEV (sieroprevalenza superiore al 40%). L'RNA di HEV è stato estratto (3), purificato con il kit NucliSENS® MiniMag (bioMérieux) e rilevato tramite RT Real-time PCR utilizzando primer specifici per la regione ORF-2 del genoma virale. La prevalenza e l’intervallo di confidenza (CI95%) sono stati calcolati con il metodo di Blaker. I ceppi virali sono stati genotipizzati secondo il protocollo dell’European network for the HEV study (HEVnet), sequenziando la regione ORF-2. Le sequenze ottenute sono state inviate al database HEVnet e depositate in GenBank. L'albero filogenetico è stato creato utilizzando il metodo Neighbor-Joining e le analisi filogenetiche sono state condotte con il software MEGA 6. RISULTATI E DISCUSSIONE Sono stati campionati 190 fegati di lepre, 416 fegati di cinghiale e 100 pool di feci di scrofa. Nelle feci è stato identificato un campione positivo a HEV (prevalenza 1%, CI95% 0.18-5.45). Nelle lepri non è stata rilevata positività, mentre sono risultati positivi 50 campioni di fegato di cinghiale (prevalenza 12.02%, CI95% 9.24-15.50). La prevalenza più alta è stata individuata nel 2018-2019 (23.81%), riducendosi fino al 1.35% nel 2020-2021, per poi aumentare al 16.13% nel 2021-2022. L'analisi filogenetica è stata svolta sui 19 campioni positivi di cinghiale risultati tipizzabili ed ha rivelato che tutte le sequenze appartengono a HEV-3, sottotipo 3a (Figura 1).
Sara Arnaboldi
added a research item
Hepatitis E virus (HEV) is the agent of the hepatitis E disease that in Europe is mainly transmitted foodborne. Human cases in EU have increased in the last years and are linked to the consumption of raw or undercooked pork product or wild boar meat infected by the zoonotic HEV-3 genotype. Rabbit HEV (HEV-3ra) is a recent genotype of HEV detected in feral, wild and farmed rabbit species (France 23%, Netherlands 60% and Germany 37.3%), and, only recently, in humans in both France and Switzerland. The genotype is now considered zoonotic, most likely affecting immunocompromises but so far, no foodborne transmission has been described. Genetically, the virus strictly resembles the zoonotic genotype HEV-3, except for an insertion in the ORF1 and more rarely in the overlapping ORF1-ORF2 region (recombinant subtype). The infection in rabbits is similar to other animal hosts and the virus is localized in several organs besides the liver. In Italy, two studies confirmed the circulation of the virus in pet and wild rabbit populations, by detection of anti-HEV IgG. The viral RNA has been detected rarely in only a few wild animals in Tuscany region and matched with both HEV-3 and HEV-3ra. In the present study, we investigated the occurrence of HEV-RNA in liver of 328 brown hares (Lepus europaeus) sampled in 13 Italian regions between 2019 and 2021, in the framework of the regional surveillance plans for wildlife diseases. HEV-RNA (0/328 liver samples) and anti-HEV antibodies (0/34 meat juice) were not detected in any tested hare. In conclusion, even if the hare seems not to be involved in the epidemiology of HEV further investigation is needed to constantly monitor the HEV presence in wild hosts, and to prevent possible foodborne zoonotic spill-over of both HEV-3 and HEV-3ra genotypes.
Sara Arnaboldi
added a research item
Torque teno sus virus (TTSuV) is a non-enveloped circular ssDNA virus which frequently infects swine and has been associated with hepatic, respiratory, and autoimmune disorders. TTSuV’s pathogenic role is still uncertain, and clear data in the literature on virus reservoirs are lacking. The aims of this study were to investigate the presence of potentially zoonotic TTSuV in wild animals in Northern Italy and to evaluate their role as reservoirs. Liver samples were collected between 2016 and 2020 during four hunting seasons from wild boars (Sus scrofa), red deer (Cervus elaphus), roe deer (Capreolus capreolus), and chamois (Rupicapra rupicapra). Samples originated from areas in Northern Italy characterized by different traits, i.e., mountains and flatland with, respectively low and high farm density and anthropization. Viral identification was carried out by end-point PCR with specific primers for TTSuV1a and TTSuVk2a species. TTSuV prevalence in wild boars was higher in the mountains than in the flatland (prevalence of 6.2% and 2.3%, respectively). In wild ruminants only TTSuVk2a was detected (with a prevalence of 9.4%). Our findings shed light on the occurrence and distribution of TTSuV in some wild animal species, investigating their possible role as reservoirs.
Sara Arnaboldi
added 2 research items
Knowledge of Mammalian orthoreovirus (MRV) and Torque teno virus (TTV) as new emerging infectious agents that may affect wild animals, is essential to minimize the risk of their spreading to farm animals and possibly to humans. The aim of this study was to investigate the presence of possibly zoonotic MRV and TTV in wild animals in Northern Italy. Our findings shed light on some aspects of occurrence and distribution of MRV and TTV in some wild species and posed the issue of their possible role as reservoir.
Aim: The aim of this study was to investigate the occurrence and distribution of Hepatitis E virus (HEV), Mammalian orthoreovirus (MRV), and Torque teno sus virus (TTSuV) in wild animals in Northern Italy to assess their potential role as zoonotic reservoirs, and to gain insight on the risk of their spreading to farm animals and possibly to humans. Methods: In this study, two different areas with different environmental features were investigated. The Sondrio area (Lombardy Alps) was characterized by mountains, low anthropization of the territory, low farm density and high conservation of natural ecosystems. The other area was near Parma, in the middle of the Po Valley, a highly anthropic area with high pig farm density. In particular, liver samples were collected between 2016 and 2020 during hunting seasons and included wild boar (Sus scrofa, n=405), red deer (Cervus elaphus, n=118), roe deer (Capreolus capreolus, n=6), chamois (Rupicapra rupicapra, n=4), and hares (Lepus europaeus, n=165). Viral identification was carried out by One-Step RT Real-time PCR for HEV, and by PCR detection for both MRV and TTSuV 1a and 1b species (genus Iotatorquevirus). Prevalence and CI95% were calculated with the Blaker’s method. Results: This study highlighted a relatively high (22.29%, CI95% 16.75-29.01) HEV prevalence in the wild boar population of the Parma area, compared to a lower prevalence in the Sondrio province (1.30%, CI95% 0.44-3.76), while no HEV positivity was found among wild ruminants or hares. In contrast, an inverse situation was found for MRV, with a prevalence in wild boars of 47.83% (CI95% 41.46-55.36) in the Sondrio area, higher than in the Parma area (15.83%, CI95% 10.83-21.52). Moreover, a 39.06% prevalence was found in wild ruminants (CI95% 31.05-47.72). TTSuV1 and 2 prevalence in wild boars were comparable both in the Parma area (1.71%, CI95% 0.58-4.92 for TTSuV1; and 1.14%, CI95%0.31-4.07 for TTSuV2), and in the Sondrio province (3.48%, CI95% 1.77-6.71). Concerning wild ruminants, a 9.38% (CI95% 5.44-15.67) prevalence of TTSuV2 was found in the Sondrio province. Furthermore, in hares TTSuV2 prevalence was as high as 37.58% (CI95% 30.55-45.17), and appears to be higher than TTSuV1 prevalence (3.03%, CI95% 1.30-6.90). Conclusions: Our findings shed light on some aspects of occurrence and distribution of HEV, MRV, and TTSuV in wild. In a global One Health perspective, the comparison of data collected both in the human, domestic, and wild animal compartments would be of the greatest importance to better understand human exposure to zoonotic viruses and the epidemiological role of different animal reservoirs.
Sara Arnaboldi
added an update
Aim: The aim of this study was to investigate the occurrence and distribution of Hepatitis E virus (HEV), Mammalian orthoreovirus (MRV), and Torque teno sus virus (TTSuV) in wild animals in Northern Italy to assess their potential role as zoonotic reservoirs, and to gain insight on the risk of their spreading to farm animals and possibly to humans.
Methods: In this study, two different areas with different environmental features were investigated. The Sondrio area (Lombardy Alps) was characterized by mountains, low anthropization of the territory, low farm density and high conservation of natural ecosystems. The other area was near Parma, in the middle of the Po Valley, a highly anthropic area with high pig farm density. In particular, liver samples were collected between 2016 and 2020 during hunting seasons and included wild boar (Sus scrofa, n=405), red deer (Cervus elaphus, n=118), roe deer (Capreolus capreolus, n=6), chamois (Rupicapra rupicapra, n=4), and hares (Lepus europaeus, n=165). Viral identification was carried out by One-Step RT Real-time PCR for HEV, and by PCR detection for both MRV and TTSuV 1a and 1b species (genus Iotatorquevirus). Prevalence and CI95% were calculated with the Blaker’s method.
Results: This study highlighted a relatively high (22.29%, CI95% 16.75-29.01) HEV prevalence in the wild boar population of the Parma area, compared to a lower prevalence in the Sondrio province (1.30%, CI95% 0.44-3.76), while no HEV positivity was found among wild ruminants or hares. In contrast, an inverse situation was found for MRV, with a prevalence in wild boars of 47.83% (CI95% 41.46-55.36) in the Sondrio area, higher than in the Parma area (15.83%, CI95% 10.83-21.52). Moreover, a 39.06% prevalence was found in wild ruminants (CI95% 31.05-47.72). TTSuV1 and 2 prevalence in wild boars were comparable both in the Parma area (1.71%, CI95% 0.58-4.92 for TTSuV1; and 1.14%, CI95%0.31-4.07 for TTSuV2), and in the Sondrio province (3.48%, CI95% 1.77-6.71). Concerning wild ruminants, a 9.38% (CI95% 5.44-15.67) prevalence of TTSuV2 was found in the Sondrio province. Furthermore, in hares TTSuV2 prevalence was as high as 37.58% (CI95% 30.55-45.17), and appears to be higher than TTSuV1 prevalence (3.03%, CI95% 1.30-6.90).
Conclusions: Our findings shed light on some aspects of occurrence and distribution of HEV, MRV, and TTSuV in wild. In a global One Health perspective, the comparison of data collected both in the human, domestic, and wild animal compartments would be of the greatest importance to better understand human exposure to zoonotic viruses and the epidemiological role of different animal reservoirs.
 
Sara Arnaboldi
added 2 research items
Hepatitis E virus (HEV) is the causative agent of hepatitis E, an emerging public health infection which has an increasing incidence across Europe. Because of the apparent lack of species barriers, HEV was characterized as a zoonotic agent. Swine are recognized as the main reservoir, but HEV is also found in wild animals such as ungulates, lagomorphs, and bats. Our work aimed at detecting the HEV presence in wild fauna in two hunting areas of Northern Italy (Parma and Sondrio areas) with different environmental and anthropic characteristics to investigate its possible role as reservoir. Liver samples were collected from wild boars, red deer, roe deer and chamois, and viral identification was carried out by One-Step RT Real-time PCR. Positive samples were genotyped, and phylogenetic analysis was performed. The virus was found only in the wild boar population, with different prevalence and subtypes in the two areas (14% HEV3a and 1.2% close to HEV3f in Parma and Sondrio, respectively). Wild ruminants seem otherwise to pose a marginal risk. Given the high pig farm density in the Parma area, and expansion of the wild boar population, continuous monitoring of the strains circulating in wildlife is crucial.
Mammalian orthoreoviruses (MRVs) are emerging infectious agents that may affect wild animals. MRVs are usually associated with asymptomatic or mild respiratory and enteric infections. However, severe clinical manifestations have been occasionally reported in human and animal hosts. An insight into their circulation is essential to minimize the risk of diffusion to farmed animals and possibly to humans. The aim of this study was to investigate the presence of likely zoonotic MRVs in wild ungulates. Liver samples were collected from wild boar, red deer, roe deer, and chamois. Samples originated from two areas (Sondrio and Parma provinces) in Northern Italy with different environmental characteristics. MRV detection was carried out by PCR; confirmation by sequencing and typing for MRV type 3, which has been frequently associated with disease in pigs, were carried out for positive samples. MRV prevalence was as high as 45.3% in wild boars and 40.6% in red deer in the Sondrio area, with lower prevalence in the Parma area (15.4% in wild boars). Our findings shed light on MRV occurrence and distribution in some wild species and posed the issue of their possible role as reservoir.
Sara Arnaboldi
added a project goal
This project evaluates the occurrence of Hepatitis E virus (HEV), Mammalian orthoreovirus (MRV), and Torque teno sus virus (TTSuV) in wild animals in Northern Italy to better estimate the risk of transmission to farmed animals and possibly to humans.