Detection of Rat Hepatitis E Virus in Pigs, Spain, 2023.

We identified rat hepatitis E virus (HEV) RNA in farmed pigs from Spain. Our results indicate that pigs might be susceptible to rat HEV and could serve as viral intermediaries between rodents and humans. Europe should evaluate the prevalence of rat HEV in farmed pigs to assess the risk to public health.

Detection and genotyping of zoonotic microsporidia in the endangered Iberian lynx (Lynx pardinus).

Microsporidia is a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most frequently reported species in humans. Limited information is available about the presence and molecular diversity of microsporidian species in the endangered Iberian lynx (Lynx pardinus). Presence of Enterocytozoon bieneusi and Encephalitozoon spp. was investigated by molecular methods in wild and captive Iberian lynxes from Spain. Overall, E. bieneusi was detected in 3.2% (8/251) of the animals examined. None of the samples tested were positive for Encephalitozoon spp. Four known (D, EbfelA, PigEBITS7, and Type IV) and a novel (named as LynxSpEb1) E. bieneusi genotypes were identified. All the genotypes found belonged to the zoonotic Group 1 of E. bieneusi. This study provides the first genotyping data of E. bieneusi in Iberian lynx in Spain. Our result indicate that the Iberian lynx does not seem to play a relevant role in the epidemiology of Encephalitozoon spp., and that this endangered felid is likely acting as spillover host rather than a true reservoir of E. bieneusi. Additional studies should be conducted to assess the impact of this parasite in the health status of the endangered Iberian lynx.

Exposure to Coxiella burnetii in Wild Lagomorphs in Spanish Mediterranean Ecosystems.

Coxiella burnetii is an important zoonotic pathogen of worldwide distribution that can infect a wide range of wild and domestic species. The European wild rabbit (Oryctolagus cuniculus) can play a role as a reservoir for this bacterium in certain epidemiological scenarios, but, to date, a very limited numbers of large-scale serosurveys have been conducted for this species worldwide. Although exposure in hare species has also been described, C. burnetii in Iberian hare (Lepus granatensis) has never been assessed. Here, we aimed to determine the seroprevalence and risk factors associated with C. burnetii exposure in wild lagomorphs in the Mediterranean ecosystems of southern Spain. Between the 2018/2019 and 2021/2022 hunting seasons, blood samples from 638 wild lagomorphs, including 471 wild rabbits and 167 Iberian hares, were collected from 112 hunting grounds distributed across all eight provinces of Andalusia (southern Spain). The overall apparent individual seroprevalence was 8.9% (57/638; 95% CI: 6.8-11.4). Antibodies against C. burnetii were found in 11.3% (53/471; 95% CI: 8.4-14.1) of the wild rabbits and 2.4% (4/167; 95% CI: 0.1-4.7) of the Iberian hares. Seropositive animals were detected for 16 (14.3%; 95% CI: 7.8-20.8) of the 112 hunting grounds tested and in all the hunting seasons sampled. A generalized estimating equations model showed that the geographical area (western Andalusia) and presence of sheep were risk factors potentially associated with C. burnetii exposure in wild lagomorphs. A statistically significant spatial cluster (p < 0.001) was identified in the south-west of Andalusia. Our results provide evidence of moderate, endemic and heterogeneous circulation of C. burnetii in wild lagomorph populations in Spanish Mediterranean ecosystems. Risk-based strategies for integrative surveillance programs should be implemented in these species to reduce the risk of transmission of the bacterium to sympatric species, including humans.

Detection and Molecular Diversity of Cryptosporidium spp. and Giardia duodenalis in the Endangered Iberian Lynx (Lynx pardinus), Spain.

Cryptosporidium spp. and Giardia duodenalis are the main non-viral causes of diarrhoea in humans and domestic animals globally. Comparatively, much less information is currently available in free-ranging carnivore species in general and in the endangered Iberian lynx (Lynx pardinus) in particular. Cryptosporidium spp. and G. duodenalis were investigated with molecular (PCR and Sanger sequencing) methods in individual faecal DNA samples of free-ranging and captive Iberian lynxes from the main population nuclei in Spain. Overall, Cryptosporidium spp. and G. duodenalis were detected in 2.4% (6/251) and 27.9% (70/251) of the animals examined, respectively. Positive animals to at least one of them were detected in each of the analysed population nuclei. The analysis of partial ssu rRNA gene sequences revealed the presence of rodent-adapted C. alticolis (n = 1) and C. occultus (n = 1), leporid-adapted C. cuniculus (n = 2), and zoonotic C. parvum (n = 2) within Cryptosporidium, and zoonotic assemblages A (n = 5) and B (n = 3) within G. duodenalis. Subgenotyping analyses allowed for the identification of genotype VaA19 in C. cuniculus (gp60 locus) and sub-assemblages AI and BIII/BIV in G. duodenalis (gdh, bg, and tpi loci). This study represents the first molecular description of Cryptosporidium spp. and G. duodenalis in the Iberian lynx in Spain. The presence of rodent/leporid-adapted Cryptosporidium species in the surveyed animals suggests spurious infections associated to the Iberian lynx's diet. The Iberian lynx seems a suitable host for zoonotic genetic variants of Cryptosporidium (C. parvum) and G. duodenalis (assemblages A and B), although the potential risk of human transmission is regarded as limited due to light parasite burdens and suspected low excretion of infective (oo)cysts to the environment by infected animals. More research should be conducted to ascertain the true impact of these protozoan parasites in the health status of the endangered Iberian lynx.

Rat hepatitis E virus (Rocahepevirus ratti) in people living with HIV.

Rat hepatitis E virus (ratHEV; species Rocahepevirus ratti) is considered a newly emerging cause of acute hepatitis of zoonotic origin. ratHEV infection of people living with HIV (PLWH) might portend a worse, as with hepatitis E virus (HEV; species Paslahepevirus balayani), and consequently this group may constitute a high-risk population. We aimed to evaluate the prevalence of ratHEV by measuring viral RNA and specific IgG antibodies in a large Spanish cohort of PLWH. Multicentre study conducted in Spain evaluating PLWHIV included in the Spanish AIDS Research Network (CoRIS). Patients were evaluated for ratHEV infection using PCR at baseline and anti-ratHEV IgG by dot blot analysis to evaluate exposure to ratHEV strains. Patients with detectable ratHEV RNA were followed-up to evaluate persistence of viremia and IgG seroconversion. Eight-hundred and forty-two individuals were tested. A total of 9 individuals showed specific IgG antibodies against ratHEV, supposing a prevalence of 1.1 (95% CI; 0.5%-2.1%). Of these, only one was reactive to HEV IgG antibodies by ELISA. One sample was positive for ratHEV RNA (prevalence of infection: 0.1%; 95% CI: 0.08%-0.7%). The case was a man who had sex with men exhibiting a slightly increased alanine transaminase level (49 IU/L) as only biochemical alteration. In the follow-up, the patients showed undetectable ratHEV RNA and seroconversion to specific ratHEV IgG antibodies. Our study shows that ratHEV is geographical broadly distributed in Spain, representing a potential zoonotic threat.

Epidemiological survey and risk factors associated with Paslahepevirus balayani in equines in Europe.

Paslahepevirus balayani (HEV) is an important emerging zoonotic virus in Europe. Although domestic pigs and wild boar are the main reservoirs of this pathogen, susceptibility to this virus has been confirmed in a growing number of animal species, including equines. However, their role in the epidemiology of this virus remains poorly understood. Our aim was to assess HEV circulation and identify potential risk factors associated with exposure in equid species in different European countries. A total of 596 equines, including 496 horses, 63 donkeys and 37 mules/hinnies bred in four European countries (Spain, Italy, United Kingdom and Ireland) were sampled. Thirty-three animals (5.5%; 95%CI: 3.7-7.4) had anti-HEV antibodies. Seropositivity was found in 4.6% of horses, 11.1% of donkeys and 8.1% of mules/hinnies tested. By country, 6.3%, 5.4%, 5.0% and 4.0% of the equines sampled in Spain, Italy, United Kingdom and Ireland, respectively, were seropositive, respectively. Statistical analysis showed that "species" and "drinking water from ponds and streams" were potential risk factors associated with HEV seropositivity in equines in Europe. HEV RNA was not detected in any (0.0%; 95%CI: 0.0-1.8) of the 202 equines tested. Our results provide evidence of a low, spatially homogeneous and widespread viral circulation that is not equal across species in equid populations in the European countries analyzed and indicate that these species appear to play a limited role in the epidemiology of this virus. Further studies are required to elucidate the differences in seroprevalence between donkeys, mules/hinnies and horses and to determine the risk of zoonotic transmission of this pathogen from equid species.

Butchering activity is the main risk factor for hepatitis E virus (Paslahepevirus balayani) infection in southwestern Nigeria: a prospective cohort study.

Introduction: Paslahepevirus balayani (Hepatitis E virus; HEV) is an emerging virus that poses as a public health threat. The virus is now reported to be the leading cause of acute viral hepatitis, with a unique impact on African settings. Our aim was to evaluate the prevalence and risk factors for HEV infection in three cohorts (animal handlers, villagers, and students). Methods: A prospective cross-sectional study was carried out on a total of 752 subjects from southwestern Nigeria. In all individuals, anti-HEV IgG and anti-HEV IgM antibodies were evaluated by using ELISA (confirming positive results via immunoblotting), and serum viral RNA was evaluated by using two RT-PCR assays. Results: The overall seroprevalence of HEV IgG and HEV IgM was 14.9% (95% CI: 12.5-17.6%) and 1.3% (95% CI: 0.7-2.5%), respectively. We observed the highest seroprevalence among animal contact individuals, with butchers being the population with the highest HEV IgG seroprevalence (31.1%). Similarly, HEV IgM was higher in the animal contact group (2.2%) than in the non-animal contact cohort (0%). Discussions: Viral RNA was not detected in any of the samples. Butchering was significantly associated with higher HEV prevalence. Although all efforts to prevent HEV in Africa have focused on the chlorination of water, our study suggests that most new infections could currently be linked to animal manipulation. Therefore, education and guidelines must be provided in southwest Nigeria to ensure that animal handling and processing methods are safe.

Lack of associations of microRNAs with severe NAFLD in people living with HIV: discovery case-control study.

Background & objective: Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in people living with HIV (PLWH) and the expression of some microRNAs could be useful as biomarkers for the diagnosis of NAFLD. The aim of this study was to identify patterns of differential expression of microRNAs in PLWH and assess their diagnostic value for NALFD. Methods: A discovery case-control study with PLWH was carried out. The expression of miRNAs was determined using HTG EdgeSeq technology. Cases were defined as patients with severe NAFLD and controls as patients without NAFLD, characterized using the controlled attenuation parameter (CAP). Cases and controls were matched 1:1 for age, sex, BMI, CD4+ lymphocyte count, active HCV infection, and ART regimen. Results: Serum 2,083 simultaneous microRNA transcripts were analyzed using HTG technology and compared between cases and controls. Forty-five patients, 23 cases, and 22 controls were included in the study. In the analysis of the expression pattern of the 2,083 microRNAs, no differential expression patterns were found between both groups of patients included in the study. Conclusion: Analysis of the microRNA transcriptome profile of nonobese PLWH with severe NAFLD did not appear to differ from that of patients without NAFLD. Thus, microRNA might not serve as a proper biomarker for predicting severe NALFD in this population.

Monitoring the epidemic of West Nile virus in equids in Spain, 2020-2021.

The largest epidemic of West Nile virus (WNV) reported ever in Spain in both humans and equines occurred in 2020, affecting 77 humans and 139 equine herds. Here, we aimed to monitor the outbreaks detected in equid herds in Andalusia (southern Spain), the Spanish region where 89.9% of the outbreaks were reported, and to evaluate the virus circulation and risk factor associated with WNV exposure in the affected herds. The first WNV case was detected in mid-July 2020, the number of outbreaks peaked in mid-August and the last one was confirmed on 26th October 2020. WNV lineage 1 was detected in 12 clinically affected horses using real time RT-PCR. Molecular analysis evidenced high nucleotide identity with WNV sequences obtained from humans, birds and mosquitoes from Spain and Italy between 2020 and 2022. Between five and eight months after the WNV epidemic, a total of 724 equids (including 485 unvaccinated and 239 vaccinated animals) from 113 of the 125 affected herds in Andalusia were sampled. IgM and IgG antibodies against WNV were detected in 1.6% (8/485; 95%IC: 0.0-2.5) and 61.9% (300/485; 95%IC: 58.3-65.5) of the unvaccinated individuals, respectively. The seropositivity in vaccinated horses was 86.6% (207/239). The main risk factors associated with WNV exposure in unvaccinated equids were the breed (crossbreed), the location of animals in spring-summer (outside), and the presence of natural water ponds close to the surveyed herds. The high individual seroprevalence obtained in the affected herds indicates that WNV circulation was more widespread than the reported by passive surveillance during the WNV epidemic in 2020. The re-emergence of WNV in 2020 in southern Spain evidenced the needed to improve integrated surveillance systems, minimizing the impact of future cases in equids and humans in high-risk areas.

Temporal changes in the genotypes of Paslahepevirus balayani in southern Spain and their possible link with changes in pig trade imports.

Introduction: Paslahepevirus balayani (HEV) is an endemic zoonotic disease ranked as a major cause of acute hepatitis in Europe. Most infections occurring in Europe are due to the endemic several subtypes of genotype 3, through the consumption of raw or undercooked pork, observing a genotype geographical distribution pattern among countries Because of global changes in the pig and pork trading markets, subtype distribution might vary. We aimed to evaluate the temporal distribution of HEV genotypes in patients from southern Spain with acute hepatitis to determine whether these changes were related to the pig import trade during the study period between 2018 and 2022. Methods: Prospective longitudinal study including patients with acute hepatitis from southern Spain between 2018 and 2022. HEV RNA and antibodies was tested in all patients. In patients with detectable HEV RNA, genotype was obtained. To determine the number of imported pigs and their origins, we checked the official data from the Spanish statistics on international trade of Spanish Minister of Industry during by country of origin during the same study period. Results: A total of 659 patients with acute hepatitis were included in the study. Among them, 162 (24.5%) had at least one marker (IgM or RNA) of acute HEV infection. Among the 71 patients with detectable viral RNA, genotypes could be obtained for 58 (81.6%). The most prevalent HEV genotype was 3f (n = 48; 78.6%), showing a decreasing prevalence of over time, from 100% in 2018 to 70.6% in 2022. Since 2021, the emergence of other genotypes has been determined. A significant increase in the number of animals imported was observed since the beginning of the study. Denmark experienced a significant rise, from 0.03% in 2018 of total imports to 10.4% in 2022. Conclusions: HEV molecular diversity is changing in Spain, could be linked to changes in fattening pig import origin.

Optimization of the molecular diagnosis of the acute hepatitis E virus infection.

To evaluate the diagnostic value of the combination of two broad-range PCR assays targeting two different and conserved regions of the viral genome for the diagnosis of acute Hepatitis E virus (HEV) infection. Patients with acute hepatitis were prospectively recruited. In all, HEV-IgM antibodies were tested together with evaluation of HEV viraemia by two PCR assays (ORF3 and ORF1). The number of individuals exhibiting negative IgM antibody results but carrying viral RNA was calculated by each PCR assay. Four-hundred and seventy individuals were included, of whom 145 (30.8%) were diagnosed as having acute HEV. Of them, 122 (84.1%) exhibited HEV-IgM antibodies, and 81 (55.8%) had detectable viral RNA for at least one PCR. Using the ORF3 molecular assay, 70 (48.3%) individuals were identified with HEV infection. When the ORF1 molecular assay was applied, 49 (33.8%) individuals were identified. The ORF3 assay detected viral RNA in 32 patients not detected by the ORF1 assay. In contrast, the ORF1 assay could amplify viral RNA in 11 patients who were not detected by the ORF3 assay. The parallel use of two broad-range PCR assays significantly increased the performance of the molecular diagnosis of HEV.

Iberian wild leporidae as hosts of zoonotic enteroparasites in Mediterranean ecosystems of Southern Spain.

Wild lagomorphs including rabbits and hares can act as natural carriers or reservoirs of bacterial and parasitic zoonotic diseases. However, little is known on the epidemiology and potential public health significance of intestinal eukaryotes in wild leporids. We examined faecal samples from European wild rabbits (Oryctolagus cuniculus, n = 438) and Iberian hares (Lepus granatensis, n = 111) collected in the Autonomous Region of Andalusia in southern Spain during 2012-2021. We searched for the presence of DNA from the main intestinal protist and microsporidial pathogens of veterinary and public health concerns using molecular methods (PCR followed by Sanger and next-generation sequencing). Giardia duodenalis was the most prevalent species found (27.8%, 153/550; 95% CI: 24.1-31.8), followed by Cryptosporidium spp. (1.3%, 7/550; 95% CI: 0.5-2.6), Blastocystis sp. (1.1%, 6/550; 95% CI: 0.4-2.4) and Encephalitozoon intestinalis (0.2%, 1/550; 95% CI: 0.0-10.1). All samples tested negative for Enterocytozoon bieneusi. Sequence analyses revealed the presence of sub-assemblage BIV (n = 1) within G. duodenalis, and Cryptosporidium cuniculus (n = 6) and Cryptosporidium andersoni (n = 1) within Cryptosporidium. The presence of ruminant-adapted C. andersoni is indicative of a potential cross-species transmission event, although a spurious infection (mechanical carriage) cannot be ruled out. Samples assigned to C. cuniculus belonged to the gp60 subtype families Va (n = 3) and Vb (n = 2). The six Blastocystis-positive samples were identified as ST2 (n = 3) and ST1 + ST2 (n = 3). Our molecular results suggest that wild rabbits and hares were primarily infected by leporid-adapted species of eukaryotic pathogens. However, the occasional findings of zoonotic G. duodenalis sub-assemblage BIV, Blastocystis sp. ST1 and ST2, and Encephalitozoon intestinalis could be of public health relevance.

Hepatitis E Virus Infections in Free-Ranging and Captive Cetaceans, Spain, 2011-2022.

Epidemiologic surveillance of hepatitis E virus in over 300 free-ranging and captive cetaceans in waters off Spain revealed extensive exposure to this pathogen. We suggest the persistent and widespread presence of hepatitis E in the marine environment off the coast of Spain may be driven by terrestrial sources of contamination.

Spatiotemporal monitoring of myxomatosis in European wild rabbit (Oryctolagus cuniculus) in Spanish Mediterranean ecosystems.

A long-term active epidemiological surveillance programme was conducted to determine seroprevalence to myxoma virus (MYXV), infection prevalence and spatiotemporal patterns and factors associated with MYXV circulation in wild rabbits (Oryctolagus cuniculus) in Spanish Mediterranean ecosystems. A total of 2376 animals were sampled over four study periods: 2009-2012 (P1), 2012-2015 (P2), 2015-2018 (P3) and 2018-2021 (P4). Antibodies against MYXV were detected by a commercial indirect ELISA in 59.9% (1424/2376; 95% CI: 58.0-61.9) of wild rabbits. At least one seropositive animal was detected on 131 (96.3%) of 136 game estates sampled. MYXV infection was confirmed by PCR in 94 of 1063 (8.8%; 95% CI: 7.3-10.7) wild rabbits. Circulation of the novel recombinant MYXV (ha-MYXV) was not found in wild rabbits analysed during P4. Five statistically significant spatiotemporal clusters of high MYXV seroprevalence were identified using a Bernoulli model: one in P2 and four in P3. A generalized linear mixed model (GLMM) analysis identified sampling season (autumn), age (adult and juvenile), outbreaks of myxomatosis in the month prior to sampling, mean annual temperature, humidity and seropositivity to rabbit haemorrhagic disease virus as factors potentially linked with MYXV seropositivity. GLMM analysis identified outbreaks of myxomatosis in the month prior to sampling, MYXV seropositivity and presence of lesions compatible with myxomatosis as factors associated with MYXV infection. The results indicate high exposure, widespread but non-homogeneous distribution, and endemic circulation of MYXV in wild rabbit populations in southern Spain during the last decade. Prevalence of antibodies against MYXV showed fluctuations both within the year and over the study periods, revealing variations in the immunity of wild rabbit populations in Mediterranean ecosystems that could increase the risk of MYXV re-emergence in immunologically naïve populations. The present study highlights the importance of long-term surveillance to better understand the epidemiology of MYXV in wild lagomorphs.

Monitoring of hepatitis E virus in zoo animals from Spain, 2007-2021.

Hepatitis E virus (HEV, family Hepeviridae) is an important emerging and zoonotic pathogen. In recent decades, the number of human cases of zoonotic hepatitis E has increased considerably in industrialized countries and HEV has been detected in an expanding range of mammal species. Although domestic pigs and wild boar are considered the main reservoirs of zoonotic HEV genotypes, the role of other susceptible animals in the epidemiology of the virus is still poorly understood. A large-scale, long-term study was carried out (1) to assess HEV exposure in captive zoo animals in Spain and (2) to determine the dynamics of seropositivity in individuals that were sampled longitudinally during the study period. Between 2007 and 2021, serum samples from 425 zoo animals belonging to 109 animal species (including artiodactyls, carnivores, perissodactyls, proboscideans and rodents) were collected from 11 different zoological parks in Spain. Forty-six of these animals at seven of these zoos were also longitudinally sampled. Anti-HEV antibodies were detected in 36 (8.5%; 95% CI: 5.8-11.1) of 425 sampled zoo animals. Specific antibodies against HEV-3 and HEV-C1 antigens were confirmed in ELISA-positive animals using western blot assay. Two of 46 longitudinally surveyed animals seroconverted during the study period. Seropositivity was significantly higher in carnivores and perissodactyls than in artiodactyls, and also during the period 2012-2016 compared with 2007-2011. HEV RNA was not detected in any of the 262 animals that could be tested by RT-PCR. To the best of the author's knowledge, this is the first large-scale, long-term surveillance on HEV in different orders of zoo mammals. Our results indicate exposure to HEV-3 and HEV-C1 in zoo animals in Spain and confirm a widespread but not homogeneous spatiotemporal circulation of HEV in captive species in this country. Further studies are required to determine the role of zoo species, particularly carnivores and perissodactyls, in the epidemiology of HEV and to clarify the origins of infection in zoological parks.

Presence of hepatitis E virus in testis of naturally infected wild boars.

The hepatitis E virus (HEV) is the main cause of viral acute hepatitis in the world, affecting more than 20 million people annually. During the acute phase of infection, HEV can be detected in various body fluids, which has a significant impact in terms of transmission, diagnosis or extrahepatic manifestations. Several studies have isolated HEV in the genitourinary tract of humans and animals, which could have important clinical and epidemiological implications. So, our main objective was to evaluate the presence of HEV in testis of naturally infected wild boars (Sus scrofa). For it, blood, liver, hepatic lymph node and testicle samples were collected from 191 male wild boars. The presence of HEV was evaluated in serum by PCR, as well as in tissues by PCR and immunohistochemistry. Four animals (2.09%; 95%CI: 0.82-5.26) showed detectable HEV RNA in serum, being confirmed the presence of HEV-3f genotype in three of them by phylogenetic analysis. HEV was also detected in liver and/or hepatic lymph nodes of the four animals by RT-PCR, as well as by immunohistochemistry analysis. Only one of these wild boars also showed detectable viral load in testis, observing HEV-specific labelling in a small number of fibroblasts and some Sertoli cells. Our results confirm the presence of HEV genotype 3 in naturally infected wild boar testis, although no associated tissue damage was evidenced. This study does not allow us to discard semen as a possible source of HEV transmission in suids. Future experimental studies are necessary to evaluate the impact of HEV genotype 3 on fertility and the possibility of transmission through sexual contact in this specie.

Hepatitis E virus in the endangered Iberian lynx (Lynx pardinus).

Hepatitis E virus (HEV) is an emerging zoonotic pathogen in Europe. In the Iberian Peninsula, wild boar (Sus scrofa) is considered the main wildlife reservoir of HEV. This wild ungulate shares habitat and resources with other potential HEV carriers in Iberian Mediterranean ecosystems, although information about the role of such sympatric species in the HEV epidemiological cycle is still very limited. The aims of the present large-scale, long-term study were: (1) to determine the seroprevalence and prevalence of HEV in both free-living and captive populations of the Iberian lynx (Lynx pardinus), the most endangered felid in the world; (2) to determine potential risk factors associated with HEV exposure in this species and (3) to evaluate the dynamics of seropositivity in longitudinally sampled animals during the study period. Between 2010 and 2021, serum samples from 275 Iberian lynxes were collected in free-ranging and captive populations across the Iberian Peninsula. Forty-four of the 275 lynxes were also longitudinally sampled during the study period. A double-antigen sandwich ELISA was used to test for the presence of antibodies against HEV. A subset of seropositive samples was analysed by Western blot (WB) assay to confirm exposure to HEV. In addition, serum, liver and/or faecal samples from 367 individuals were tested for orthohepevirus RNA by RT-PCR. A total of 50 (18.2%; 95% CI: 14.1-23.2) of the 275 animals analysed had anti-HEV antibodies by ELISA. Exposure to HEV was confirmed by WB in most of the ELISA-positive Iberian lynxes analysed. Significantly higher seroprevalence was found in captive (33.6%) compared to free-ranging (7.4%) individuals. Within captive population, the GEE model identified 'age' (senile, adult and subadult) as risk a factor potentially associated with HEV exposure in the Iberian lynx. Thirteen (29.5%) of 44 longitudinally surveyed individuals seroconverted against HEV during the study period. HEV RNA was detected in the faeces of one (1/364; 0.3%; 95% CI: 0.0-0.8) free-ranging adult animal sampled in 2021. Phylogenetic analysis showed that the sequenced strain belongs to HEV-3f subtype and shared a high nucleotide sequence identity (97-99.6%) with human HEV-3f sequences from Spain and France. To the best of the authors' knowledge, this is the first survey study on HEV in the Iberian lynx and the first molecular report of HEV-A infection in free-ranging felines. Our results indicate high exposure to HEV-3 in Iberian lynx populations, particularly those kept in captivity. The serological results suggest widespread but not homogeneous circulation of HEV in Iberian lynx populations. Further studies are required to assess the epidemiological role of this endangered species as a potential spillover host of HEV.

Epidemiological survey and risk factors associated with hepatitis E virus in small ruminants in southern Spain.

Autochthonous cases of hepatitis E (HE) associated with zoonotic genotypes HEV-3 and HEV-4 have significantly increased in industrialized countries over the last decade. Suidae are generally recognized as the main reservoirs of these genotypes. Susceptibility to HE virus (HEV) infection and zoonotic potential have also been confirmed in other species, including sheep and goat. However, the information about their role in the epidemiology of HEV remains very scarce. The objective of this study was to assess the prevalence, spatial distribution and risk factors associated with HEV exposure in sheep and goats in southern Spain, the country with the highest census of small domestic ruminants in the European Union. Blood samples from 240 sheep and 240 goats were collected between 2015 and 2017. Sera were analysed in parallel using a commercial double-antigen ELISA and real-time PCR. A total of 38 (7.9%; 95%CI: 5.5-10.3) out of 480 sampled animals showed anti-HEV antibodies. By species, the seroprevalences found in sheep and goats were 2.1% (5/240; 95%CI: 0.3-3.9) and 13.8% (33/240; 95%CI: 9.4-18.1) respectively. Anti-HEV antibodies were found on 19 (59.4%; 95%CI: 42.4-76.4) of the 32 sampled farms. The GEE model showed that species (goat) and number of small ruminants in the farm (≤348 animals and ≥538 animals) were risk factors potentially associated with HEV exposure in small ruminants in the study area. HEV RNA was not detected in any of the 480 (0.0%; 95%CI: 0.0-0.8) tested animals. Our results confirm that sheep and goats are naturally, but not equally exposed to HEV and indicate the widespread spatial distribution of HEV among small ruminant populations in southern Spain. Further studies are required to elucidate the role of sheep and goat in the epidemiology of HEV and their potential implications for public health.

Seroreversion of IgG anti-HEV in HIV cirrhotic patients: A long-term multi-sampling longitudinal study.

The aim of our study was to evaluate HEV antibody kinetics in HIV/HCV-coinfected patients with cirrhosis. A longitudinal retrospective study was designed. Patients were followed up every 6 months; anti-HEV IgG and IgM antibodies levels and HEV-RNA by qPCR were analysed. The prevalence and incidence of every HEV infection marker were calculated. The kinetics of anti-HEV IgG and IgM during the follow-up were evaluated. Seventy-five patients comprised the study population. The seroprevalence observed was 17.3%. None showed IgM antibodies or HEV-RNA at baseline. None showed detectable HEV viral load during the study period. After a median follow-up of 5.1 years, two of 62 seronegative patients (3.2%) seroconverted to IgG antibody. The incidence for IgM was 2.7%. Of the 13 patients with IgG seropositivity at baseline, five (38.5%) seroreverted. Meanwhile, of the two patients who exhibited IgM positivity during the study, one (50%) showed intermittent positivity. We found that HEV seropositivity is common in HIV/HCV-coinfected cirrhotic patients. A remarkable rate of IgG seroreversions and IgM intermittence was found, limiting the use of antibodies for the diagnosis of HEV infection in this population.

Serological and molecular survey of hepatitis E virus in cats and dogs in Spain.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen that is currently recognized as one of the major causes of acute human hepatitis worldwide. In Europe, the increasing number of hepatitis E cases is mainly associated with the consumption of animal food products or contact with infected animals. Dogs and cats have been suggested as a zoonotic source of HEV infection. The aim of this study was to assess Orthohepevirus circulation, including HEV-A, HEV-B and HEV-C species, in sympatric urban cats and dogs in southern Spain. Between 2017 and 2020, blood samples were collected from 144 stray cats and 152 dogs, both strays and pets. The presence of antibodies against HEV were tested using a double-antigen sandwich ELISA and seropositive samples were further analysed by western blot. A RT-PCR was performed to detect RNA of Orthohepevirus species (HEV-A, HEV-B and HEV-C). A total of 19 (6.4%; 95%CI: 3.6-9.2) of the 296 animals tested showed anti-HEV antibodies by ELISA. Seropositivity was significantly higher in dogs (9.9%; 15/152; 95%CI: 5.1-14.6) than in cats (2.8%; 4/144; 95%CI: 0.1-5.5). Ten of the 18 ELISA-positive animals that could be further analysed by western blot, reacted against HEV-3 and/or HEV-C1 antigens, which suggest circulation of both genotypes in urban cats and dogs in the study area. However, HEV-A, HEV-B and HEV-C RNA were not detected in any of the tested sera. This is the first study to assess HEV circulation in both stray cats and dogs in Europe. Our results provide evidence of HEV exposure in sympatric urban cat and dog populations in southern Spain. Further studies are needed to determine the role of these species in the epidemiology of HEV.