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Hepatitis E virus (HEV), species Paslahepevirus balayani, poses a global public health threat, especially in developing countries, by causing acute enterically transmitted hepatitis. HEV infects various mammalian hosts and belongs to the genus Paslahepevirus in the family Hepeviridae. While swine are recognized as the main hosts of HEV, rabbits, which can also be affected by swine HEV-3 related strains, serve as the primary reservoir for the distinct emerging and zoonotic HEV-3ra subtype. In Portugal, where the European wild rabbit is abundant, their role in HEV epidemiology remains unclear. The primary aim of the present research was to evaluate the circulation and the potential for HEV infection within these species. This study employed a molecular and longitudinal serological approach to investigate HEV in Portuguese rabbits. Among the 205 wild rabbits tested, a seroprevalence of 2.44% (95% CI: 0.80-5.60) was found, with no significant associations with age, sex, localization, or sampling dates. Seropositive animals were found in the south and center regions of the country. HEV RNA was not detected in 120 fecal samples, suggesting a natural, low level, and widespread viral circulation. The study underscores the need for further research to comprehend HEV dynamics in these species, which is crucial for assessing potential transmission risks to humans., (© 2024. The Author(s).)

Passive surveillance can be most effective in the early detection of disease outbreaks given that farmers observe their animals daily. The European Animal Health Law states that unexplained excess mortality should be reported to the veterinary authorities. In the Netherlands, in addition to notifications to the competent authority, Royal GD is commissioned a passive surveillance component that consists of a veterinary helpdesk and postmortem examination for early detection of emerging diseases. The aim of this study was to evaluate this voluntary passive surveillance component through excess mortality in cattle. Weekly on-farm mortality was calculated using the cattle Identification and Registration records. Mortality was assessed on regional level for dairy, veal and other beef cattle using a Generalized Linear Model (GLM) (log-link, negative binomial). We used a cumulative sum of the model residuals to identify periods of excess mortality. The mortality was defined as excessive when above five times the standard error. The analysis was also conducted on herd level, but these models did not converge. We checked for an association between the two passive surveillance components elements and excess mortality. A GLM (log-link, negative binomial) with the number of contacts or submissions per region as the dependent variables and excess mortality per region and year as independent variables was carried out. Overall, the models showed significantly higher use of passive surveillance components in periods of excess mortality compared to non-excess periods. In dairy cattle the odds for contact or submission were between 1.72 (1.59-1.86) and 2.02 (1.82-2.25). For veal calves we found the odds of 2.19 (1.18-4.04) and 2.24 (1.78-2.83) relative to periods without excess mortality. Beef cattle operations, other than veal, showed only an increased odds for postmortem submissions in calves of 3.71 (2.74-5.01), submissions for cattle and contact in general was not increased for this farm type. In conclusion, the voluntary passive surveillance component in the Netherlands is used more often in periods of excess mortality in cattle. The chance of getting a timely response is highest for dairy farms. For veal calf operations the chance of receiving a timely response is more likely for postmortem submissions. A comparison with passive surveillance for excess mortality in other countries was not possible because no literature could be found. However, the method of this study can be used by other countries to evaluate their passive surveillance. This would make comparison of the performance of passive surveillance in different countries possible., Competing Interests: Declaration of Competing Interest G.S. is parttime employed at Royal GD; the other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatitis E virus (HEV) is currently recognized as an emerging problem and a growing concern for public health in developed countries, with HEV infections mainly attributable to foodborne transmission of HEV-3. The zoonotic HEV genotype 3 infects a wide range of mammalian hosts, with swine considered as the primary host. This study investigates the occurrence of HEV among small ruminants in Portugal. The primary aim of the present research was to evaluate the circulation and the potential for HEV infection among sheep and goats. A total of 400 bile samples and 493 blood samples were collected from sheep and goats at a slaughterhouse in the center region of Portugal, between January 2022 and March 2023. The HEV RNA detection in bile samples was performed using a nested broad-spectrum RT-PCR targeting the ORF1 region. Serological analysis to detect anti-HEV antibodies was conducted using a commercial double-antigen sandwich multi-species ELISA. The HEV RNA was not detected in any bile samples using the nested broad-spectrum RT-PCR. Serological analysis revealed an overall HEV antibody seroprevalence of 2% (10/493, 95% CI: 0.98-3.70) among the small ruminants, namely 2.2% in goats and 2.0% in sheep. Curiously, no statistically significant association among the factors, age, sex and species and HEV seroprevalence was observed. Although HEV RNA was not detected in the bile of sheep and goats, this study the evidence of seroprevalence in these small ruminant species. Further research could provide additional insights into the factors influencing HEV transmission dynamics in small ruminants in Portugal and its potential implications for public health., (© 2024. The Author(s).)

The aim of this study was to investigate to what extent fragments of the HEV genome could be used for accurate diagnostics and inference of viral population-scale processes. For this, we selected all the published whole genome sequences from the NCBI GenBank and trimmed them to various fragment lengths (ORF1,2,3, ORF1, ORF2, ORF3, 493 nt in ORF2 and 148 nt in ORF2). Each of the fragment lengths was used to infer the richness and diversity of the viral sequence types, typing accuracy, and potential use in phylodynamics. The results obtained from the different fragments were compared. We observed that, generally, the longer the nucleic acid fragment used in typing, the better the accuracy in predicting the viral subtype. However, the dominant HEV subtypes circulating in Europe were relatively well classified even by the 493 nt fragment, with false negative rates as low as 8 in 1000 typed sequences. Most fragments also give comparable results in analyses of population size, albeit with shorter fragments showing a broader 95 % highest posterior density interval and less obvious increase of the viral effective population size. The reconstructed phylogenies of a heterochronous subset indicated a good concordance between all the fragments, with the major clades following similar branching patterns. Furthermore, we have used the HEV sequence data from the Netherlands available in the HEVnet database as a case study for reconstruction of population size changes in the past decades. This data showed that molecular and epidemiological results are concordant and point to an increase in the viral effective population size underlying the observed increase in incidence of acute HEV infection cases. In the absence of whole genome sequencing data, the 493 bp fragment can be used for analyzing HEV strains currently circulating in Europe, as it is informative for describing short term population-scale processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Urban areas are unique ecosystems with stark differences in species abundance and composition compared with natural ecosystems. These differences can affect pathogen transmission dynamics, thereby altering zoonotic pathogen prevalence and diversity. In this study, we screened small mammals from natural and urban areas in the Netherlands for up to 19 zoonotic pathogens, including viruses, bacteria, and protozoan parasites. Materials and Methods: In total, 578 small mammals were captured, including wood mice ( Apodemus sylvaticus ), bank voles ( Myodes glareolus ), yellow-necked mice ( Apodemus flavicollis ), house mice ( Mus musculus ), common voles ( Microtus arvalis ), and greater white-toothed shrews ( Crocidura russula ). We detected a wide variety of zoonotic pathogens in small mammals from both urban and natural areas. For a subset of these pathogens, in wood mice and bank voles, we then tested whether pathogen prevalence and diversity were associated with habitat type ( i.e., natural versus urban), degree of greenness, and various host characteristics. Results: The prevalence of tick-borne zoonotic pathogens ( Borrelia spp. and Neoehrlichia mikurensis ) was significantly higher in wood mice from natural areas. In contrast, the prevalence of Bartonella spp. was higher in wood mice from urban areas, but this difference was not statistically significant. Pathogen diversity was higher in bank voles from natural habitats and increased with body weight for both rodent species, although this relationship depended on sex for bank voles. In addition, we detected methicillin-resistant Staphylococcus aureus , extended-spectrum beta-lactamase/AmpC-producing Escherichia coli , and lymphocytic choriomeningitis virus for the first time in rodents in the Netherlands. Discussion: The differences between natural and urban areas are likely related to differences in the abundance and diversity of arthropod vectors and vertebrate community composition. With increasing environmental encroachment and changes in urban land use ( e.g., urban greening), it is important to better understand transmission dynamics of zoonotic pathogens in urban environments to reduce potential disease risks for public health.

West Nile virus (WNV) was first detected in the Netherlands in 2020, with circulation observed in birds, mosquitoes, and humans in two geographical areas. Usutu virus (USUV) has been circulating in the Netherlands since 2016. Following the detection of WNV in the Netherlands, we investigated the possible use of petting zoos as urban sentinel sites to examine the extent of WNV and USUV circulation around the two WNV outbreak locations. Chickens at petting zoos and in backyards were sampled within a 15-kilometer radius of the confirmed WNV circulation areas at three timepoints over one year (2021-2022). Sera were analysed using a protein microarray for binding antibodies to orthoflavivirus NS1 antigens and reactive samples were confirmed through micro-focus reduction neutralization tests (mFRNT). Furthermore, mosquitoes at sampling locations were collected to assess their blood feeding behaviour. This serosurvey detected the circulation of USUV and WNV in petting zoo and backyard chickens in 2021, both within and outside the 2020 outbreak areas. The WNV circulation was not detected by other existing surveillance schemes in mosquitoes, wild birds, horses and humans. In addition, the results show rapid decay of USUV antibodies in approximately 20 weeks. Our findings support the utility and the added value of petting zoo chickens as sentinels for monitoring USUV and WNV circulation compared to other available methods. Seroconversions observed in petting zoos and backyard chickens living in or near densely populated urban areas further highlighted potential public health risks that went undetected.

Multiple genotypes of highly pathogenic avian influenza (HPAI) H5 clade 2.3.4.4b viruses have caused epizootics in wild birds and poultry. The HPAI H5N1 genotype C virus caused a modest epizootic, whereas the occurrence of the HPAI H5N1 genotype AB virus in 2021 resulted in the largest avian influenza epizootic in Europe to date. Here we studied the pathogenicity of two HPAI H5N1 viruses by experimentally infecting chickens, Pekin ducks, Eurasian wigeons and Barnacle geese. Our study demonstrates that pathogenicity of the H5N1-2021-AB virus is lower in Pekin ducks, Eurasian wigeons and Barnacle geese compared to the H5N1-2020-C virus, whereas virus shedding was high for both viruses. After inoculation with H5N1-2021-C viral antigen expression was higher in the brain of Pekin ducks, Eurasian wigeons and Barnacle geese, which caused higher mortality compared to inoculation with H5N1-2021-AB virus. Subclinical infections occurred in Pekin ducks and Eurasian wigeons and mortality was reduced in Barnacle geese after inoculation with H5N1-2021-AB virus while H5N1-2020-C virus caused high morbidity and mortality in these species. This H5N1-2021-AB virus trait may have contributed to efficient spread of the virus in wild bird populations. Therefore, high mortality, virus shedding and long-lasting viral antigen expression found in Barnacle geese may have increased the risk for introduction into poultry.

Aims: Zoonotic arboviruses (arthropod-borne) of the Orthoflavivirus genus, such as West Nile virus (WNV), Usutu virus (USUV) and Tick-borne encephalitis virus (TBEV), are emerging in Northwestern Europe and pose a threat to both human and animal health. In the Netherlands, passive symptomatic surveillance (notification of clinical cases) in horses is one of the main pillars for the early detection of WNV. For such passive surveillance to work properly, horse owners and veterinarians need to recognize symptoms and report suspected cases to the authorities. Currently, little is known about the seroprevalence of orthoflaviviruses in domestic animals in the Netherlands. Therefore, this study aims at identifying the seroprevalence of WNV and USUV in horses and dogs in the Netherlands. Additionally, this study seeks to evaluate the knowledge and perceptions of Dutch horse owners towards mosquito-borne viruses., Methods and Results: A cross-sectional serosurvey in horses and dogs was conducted between May 2021 and May 2022. Serum samples were screened using an ELISA and doubtful and positive samples were confirmed by Virus Neutralization Tests for WNV, USUV and TBEV. A validated questionnaire, the MosquitoWise survey, was used to assess the knowledge and perceptions of Dutch horse owners towards mosquito-borne viruses between July and October 2022. The serosurvey revealed a low seroprevalence for WNV in horses and no WNV-positive dogs were found. Similarly, a low USUV seroprevalence was found in dogs. The MosquitoWise survey revealed a high knowledge level for horse owners and high awareness of WNV vaccination but a more limited intent to vaccinate., Conclusions: The low seroprevalences of WNV and USUV indicate many dogs and horses remain susceptible, offering opportunities for trend analysis and surveillance. However, despite multiple recent detections of WNV, USUV, and TBEV in humans, the role of dogs and horses in early detection of human cases is debatable. High awareness among horse owners and the absence of detected equine WNV cases highlight this uncertainty. Continued surveillance is crucial for detecting increased virus circulation and protecting both animal and human health., (© 2024 The Author(s). Zoonoses and Public Health published by Wiley‐VCH GmbH.)

The application of a One Health approach recognizes that human health, animal health, plant health and ecosystem health are intrinsically connected. Tackling complex challenges associated with foodborne zoonoses, antimicrobial resistance, and emerging threats is imperative. Therefore, the One Health European Joint Programme was established within the European Union research programme Horizon 2020. The One Health European Joint Programme activities were based on the development and harmonization of a One Health science-based framework in the European Union (EU) and involved public health, animal health and food safety institutes from almost all EU Member States, the UK and Norway, thus strengthening the cooperation between public, medical and veterinary organizations in Europe. Activities including 24 joint research projects, 6 joint integrative projects and 17 PhD projects, and a multicountry simulation exercise facilitated harmonization of laboratory methods and surveillance, and improved tools for risk assessment. The provision of sustainable solutions is integral to a One Health approach. To ensure the legacy of the work of the One Health European Joint Programme, focus was on strategic communication and dissemination of the outputs and engagement of stakeholders at the national, European and international levels.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for disease outbreaks in wild birds and poultry, resulting in devastating losses to the poultry sector. Since 2020, an increasing number of outbreaks of HPAI H5N1 was seen in wild birds. Infections in mammals have become more common, in most cases in carnivores after direct contact with infected birds. Although ruminants were previously not considered a host species for HPAI viruses, in March 2024 multiple outbreaks of HPAI H5N1 were detected in goats and cattle in the United States. Here, we have used primary bronchus-derived well-differentiated bovine airway epithelial cells (WD-AECs) cultured at air-liquid interface to assess the susceptibility and permissiveness of bovine epithelial cells to infection with European H5N1 virus isolates. We inoculated bovine WD-AECs with three low-passage HPAI clade 2.3.4.4b H5N1 virus isolates and detected rapid increases in viral genome loads and infectious virus during the first 24 h post-inoculation, without substantial cytopathogenic effects. Three days post-inoculation infected cells were still detectable by immunofluorescent staining. These data indicate that multiple lineages of HPAI H5N1 may have the propensity to infect the respiratory tract of cattle and support extension of avian influenza surveillance efforts to ruminants. Furthermore, this study underscores the benefit of WD-AEC cultures for pandemic preparedness by providing a rapid and animal-free assessment of the host range of an emerging pathogen.

Virus monitoring in small mammals is central to the design of epidemiological control strategies for rodent-borne zoonotic viruses. Synanthropic small mammals are versatile and may be potential carriers of several microbial agents. In the present work, a total of 330 fecal samples of small mammals were collected at two sites in the North of Portugal and screened for zoonotic hepatitis E virus (HEV, species Paslahepevirus balayani). Synanthropic small mammal samples (n = 40) were collected in a city park of Porto and belonged to the species Algerian mouse (Mus spretus) (n = 26) and to the greater white-toothed shrew (Crocidura russula) (n = 14). Furthermore, additional samples were collected in the Northeast region of Portugal and included Algerian mouse (n = 48), greater white-toothed shrew (n = 47), wood mouse (Apodemus sylvaticus) (n = 43), southwestern water vole (Arvicola sapidus) (n = 52), Cabrera's vole (Microtus cabrerae) (n = 49) and Lusitanian pine vole (Microtus lusitanicus) (n = 51). A nested RT-PCR targeting a part of open reading frame (ORF) 2 region of the HEV genome was used followed by sequencing and phylogenetic analysis. HEV RNA was detected in one fecal sample (0.3%; 95% confidence interval, CI: 0.01-1.68) from a synanthropic Algerian mouse that was genotyped as HEV-3, subgenotype 3e. This is the first study reporting the detection of HEV-3 in a synanthropic rodent, the Algerian mouse. The identified HEV isolate is probably the outcome of either a spill-over infection from domestic pigs or wild boars, or the result of passive viral transit through the intestinal tract. This finding reinforces the importance in the surveillance of novel potential hosts for HEV with a particular emphasis on synanthropic animals., (© 2024. The Author(s).)

Highly pathogenic avian influenza (HPAI) H5-viruses are circulating in wild birds and are repeatedly introduced to poultry causing outbreaks in the Netherlands since 2014. The largest epizootic ever recorded in Europe was caused by HPAI H5N1 clade 2.3.4.4b viruses in the period 2021-2022. The recent H5-clade 2.3.4.4 viruses were found to differ in their virulence for chickens and ducks. Viruses causing only mild disease may remain undetected, increasing the risk of virus spread to other farms, wild birds and mammals. We developed in ovo models to determine the virulence of HPAI viruses for chickens and ducks, which are fast and have low costs. The virulence of five contemporary H5-viruses was compared studying replication rate, average time to death and virus spread in the embryo. Remarkable differences in virulence were observed between H5-viruses and between poultry species. The H5N1-2021 virus was found to have a fast replication rate in both the chicken and duck in ovo models, but a slower systemic virus dissemination compared to three other H5-clade 2.3.4.4b viruses. The results show the potential of in ovo models to quickly determine the virulence of novel HPAI viruses, and study potential virulence factors which can help to better guide the surveillance in poultry.

Panels of pre- and post-pandemic farm animals, wild boar and human sera, including human sera able to neutralize SARS-CoV-2 in vitro, were tested in serological tests to determine their cross-reactivity with β- and α-CoV originating from farm animals. Sera were tested in neutralization assays with high ascending concentrations (up to 1 × 10 4 TCID 50 units/well) of β-CoV Bovine coronavirus (BCV), SARS-CoV-2, and porcine α-CoV-transmissible gastroenteritis virus (TGEV). In addition, sera were tested for immunostaining of cells infected with β-CoV porcine hemagglutinating encephalomyelitis (PHEV). Testing revealed a significantly higher percentage of BCV neutralization (78%) for sera of humans that had experienced a SARS-CoV-2 infection (SARS-CoV-2 convalescent sera) than was observed for human pre-pandemic sera (37%). Also, 46% of these human SARS-CoV-2 convalescent sera neutralized the highest concentration of BCV (5 × 10 3 TCID 50 /well) tested, whereas only 9.6% of the pre-pandemic sera did. Largely similar percentages were observed for staining of PHEV-infected cells by these panels of human sera. Furthermore, post-pandemic sera collected from wild boars living near a densely populated area in The Netherlands also showed a higher percentage (43%) and stronger BCV neutralization than was observed for pre-pandemic sera from this area (21%) and for pre- (28%) and post-pandemic (20%) sera collected from wild boars living in a nature reserve park with limited access for the public. High percentages of BCV neutralization were observed for pre- and post-pandemic sera of cows (100%), pigs (up to 45%), sheep (36%) and rabbits (60%). However, this cross-neutralization was restricted to sera collected from specific herds or farms. TGEV was neutralized only by sera of pigs (68%) and a few wild boar sera (4.6%). None of the BCV and PHEV cross-reacting human pre-pandemic, wild boar and farm animal sera effectively neutralized SARS-CoV-2 in vitro. Preexisting antibodies in human sera effectively neutralized the animal β-CoV BCV in vitro. This cross-neutralization was boosted after humans had experienced a SARS-CoV-2 infection, indicating that SARS-CoV-2 activated a "memory" antibody response against structurally related epitopes expressed on the surface of a broad range of heterologous CoV, including β-CoV isolated from farm animals. Further research is needed to elucidate if a symptomless infection or environmental exposure to SARS-CoV-2 or another β-CoV also triggers such a "memory" antibody response in wild boars and other free-living animals.

Urban greening has benefits for both human and environmental health. However, urban greening might also have negative effects as the abundance of wild rats, which can host and spread a great diversity of zoonotic pathogens, increases with urban greenness. Studies on the effect of urban greening on rat-borne zoonotic pathogens are currently unavailable. Therefore, we investigated how urban greenness is associated with rat-borne zoonotic pathogen prevalence and diversity, and translated this to human disease hazard. We screened 412 wild rats (Rattus norvegicus and Rattus rattus) from three cities in the Netherlands for 18 different zoonotic pathogens: Bartonella spp., Leptospira spp., Borrelia spp., Rickettsia spp., Anaplasma phagocytophilum, Neoehrlichia mikurensis, Spiroplasma spp., Streptobacillus moniliformis, Coxiella burnetii, Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL)/AmpC-producing Escherichia coli, rat hepatitis E virus (ratHEV), Seoul orthohantavirus, Cowpox virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Toxoplasma gondii and Babesia spp. We modelled the relationships between pathogen prevalence and diversity and urban greenness. We detected 13 different zoonotic pathogens. Rats from greener urban areas had a significantly higher prevalence of Bartonella spp. and Borrelia spp., and a significantly lower prevalence of ESBL/AmpC-producing E. coli and ratHEV. Rat age was positively correlated with pathogen diversity while greenness was not related to pathogen diversity. Additionally, Bartonella spp. occurrence was positively correlated with that of Leptospira spp., Borrelia spp. and Rickettsia spp., and Borrelia spp. occurrence was also positively correlated with that of Rickettsia spp. Our results show an increased rat-borne zoonotic disease hazard in greener urban areas, which for most pathogens was driven by the increase in rat abundance rather than pathogen prevalence. This highlights the importance of keeping rat densities low and investigating the effects of urban greening on the exposure to zoonotic pathogens in order to make informed decisions and to take appropriate countermeasures preventing zoonotic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatitis E virus (HEV) is a positive-sense single-stranded RNA virus and a major cause of acute viral hepatitis. HEV is responsible for 20 million infections worldwide in humans every year. HEV-3 and HEV-4 are zoonotic and are responsible for most of the HEV cases in developed countries. Consumption of contaminated pig meat or pig products is considered to be the main transmission route of HEV HEV-3 in Europe. Prevalence studies for HEV generally use PCR methods to detect the presence or absence of genomic RNA. However, these methods do not discriminate infectious virus particles from non-infectious material. Previously developed HEV cell culture systems only worked with high efficiency after cell line adaptation of the subjected virus strains. In this manuscript, the development of a culture system for the detection of infectious HEV strains is described. For this purpose, we optimized the isolation and the growth of primary hepatocytes from young piglets. Subsequently, the isolated hepatocytes were used to culture HEV of different origins, such as liver tissue samples and sausage samples. This method can be applied to better assess the risk of infection through consumption of food products associated with HEV RNA contamination.

The susceptibility of domestic cats to infection with SARS-CoV-2 has been demonstrated by several experimental studies and field observations. We performed an extensive study to further characterize the transmission of SARS-CoV-2 between cats, through both direct and indirect contact. To that end, we estimated the transmission rate parameter and the decay parameter for infectivity in the environment. Using four groups of pair-transmission experiment, all donor (inoculated) cats became infected, shed virus, and seroconverted, while three out of four direct contact cats got infected, shed virus, and two of those seroconverted. One out of eight cats exposed to a SARS-CoV-2-contaminated environment became infected but did not seroconvert. Statistical analysis of the transmission data gives a reproduction number R 0 of 2.18 (95% CI = 0.92 to 4.08), a transmission rate parameter β of 0.23 day -1 (95% CI = 0.06 to 0.54), and a virus decay rate parameter μ of 2.73 day -1 (95% CI = 0.77 to 15.82). These data indicate that transmission between cats is efficient and can be sustained ( R 0  > 1), however, the infectiousness of a contaminated environment decays rapidly (mean duration of infectiousness 1/2.73 days). Despite this, infections of cats via exposure to a SARS-CoV-2-contaminated environment cannot be discounted if cats are exposed shortly after contamination. IMPORTANCE This article provides additional insight into the risk of infection that could arise from cats infected with SARS-CoV-2 by using epidemiological models to determine transmission parameters. Considering that transmission parameters are not always provided in the literature describing transmission experiments in animals, we demonstrate that mathematical analysis of experimental data is crucial to estimate the likelihood of transmission. This article is also relevant to animal health professionals and authorities involved in risk assessments for zoonotic spill-overs of SARS-CoV-2. Last but not least, the mathematical models to calculate transmission parameters are applicable to analyze the experimental transmission of other pathogens between animals., Competing Interests: The authors declare no conflict of interest.

Hepatitis E virus (HEV) can be transmitted from pigs to humans and cause liver inflammation. Pigs are a major reservoir of HEV and most slaughter pigs show evidence of infection by presence of antibodies (ELISA) or viral RNA (PCR). Reducing the number of HEV infected pigs at slaughter would likely reduce human exposure, yet how this can be achieved, is unknown. We aimed to identify and quantify the effect of biosecurity measures to deliver HEV negative batches of pigs to slaughter. A case-control study was performed with Dutch pig farms selected based on results of multiple slaughter batches. Case farms delivered at least one PCR and ELISA negative batch to slaughter (PCR - ELISA - ), indicating absence of HEV infection, and control farms had the highest proportion of PCR and/or ELISA positive batches (PCR + ELISA + ), indicating high within-farm transmission. Data about biosecurity and housing were collected via a questionnaire and an audit. Variables were selected by regularization (LASSO regression) and ranked, based the frequency of variable selection. The odds ratios (OR) for the relation between case-control status and the highest ranked variables were determined via grouped logistic regression. Thirty-five case farms, with 10 to 60% PCR - ELISA - batches, and 38 control farms with on average 40% PCR + ELISA + batches, were included. Rubber and steel floor material in fattening pens had the highest ranking and increased the odds of a PCR - ELISA - batch by 5.87 (95%CI 3.03-11.6) and 7.13 (95%CI 3.05-16.9) respectively. Cleaning pig driving boards weekly (OR 1.99 (95%CI 1.07-3.80)), and fly control with predatory flies (OR 4.52 (95%CI 1.59-13.5)) were protective, whereas a long fattening period was a risk. This study shows that cleaning and cleanability of floors and fomites and adequate fly control are measures to consider for HEV control in infected farms. Yet, intervention studies are needed to confirm the robustness of these outcomes., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Author(s).)

Hepatitis E virus (HEV) represents a major cause of acute hepatitis and is considered an emerging public health problem around the world. In the Middle East's and Africa's arid regions, where camels frequently interact with human populations and camel-derived food products are a component of the food chain, camel-borne zoonotic HEV infection is a potential threat. To date, no review paper has been published on HEV in camels. As such, the purpose of the current work is to provide a scientific review of the identification of HEV genotypes seven and eight in camels worldwide to have a better understanding of the current status of this topic and to identify gaps in the current knowledge. Searches were carried out in the electronic databases PubMed, Mendeley, Web of Science, and Scopus, including studies published until 31 December 2022 ( n = 435). Once the databases were checked for duplicate papers ( n = 307), the exclusion criteria were applied to remove any research that was not relevant ( n = 118). As a result, only 10 papers were found to be eligible for the study. Additionally, in eight of the ten studies, the rates of HEV infection were found to be between 0.6% and 2.2% in both stool and serum samples. Furthermore, four studies detected HEV genotype seven in dromedary camels, and two studies have shown HEV genotype eight in Bactrian camels. Interestingly, these genotypes were recently reported in camels from the Middle East and China, where one human infection with HEV genotype seven has been associated with the consumption of contaminated camel meat and milk. In conclusion, more research will be needed to determine the prevalence of HEV infection in camels around the world as well as the risk of foodborne transmission of contaminated camel products. As camels are utility animals in several countries, HEV in these animals may pose a potential risk to public health.

Several reports demonstrated the susceptibility of domestic cats to SARS-CoV-2 infection. Here, we describe a thorough investigation of the immune responses in cats after experimental SARS-CoV-2 inoculation, along with the characterization of infection kinetics and pathological lesions. Specific pathogen-free domestic cats ( n = 12) were intranasally inoculated with SARS-CoV-2 and subsequently sacrificed on DPI (days post-inoculation) 2, 4, 7 and 14. None of the infected cats developed clinical signs. Only mild histopathologic lung changes associated with virus antigen expression were observed mainly on DPI 4 and 7. Viral RNA was present until DPI 7, predominantly in nasal and throat swabs. The infectious virus could be isolated from the nose, trachea and lungs until DPI 7. In the swab samples, no biologically relevant SARS-CoV-2 mutations were observed over time. From DPI 7 onwards, all cats developed a humoral immune response. The cellular immune responses were limited to DPI 7. Cats showed an increase in CD8+ cells, and the subsequent RNA sequence analysis of CD4+ and CD8+ subsets revealed a prominent upregulation of antiviral and inflammatory genes on DPI 2. In conclusion, infected domestic cats developed a strong antiviral response and cleared the virus within the first week after infection without overt clinical signs and relevant virus mutations., Competing Interests: The authors declare no conflicts of interest.

Swine are widely recognized as the main reservoir of zoonotic HEV; however, a growing body of data on the HEV prevalence in farmed ruminants of different species also points to a potential route for HEV transmission through ruminants and ruminant products and by-products. Definite information on the zoonotic potential of ruminants is still absent or unclear, compelling the necessity for increasing knowledge on this. The aim of the current study was to analyze the state-of-the-art in this research topic and provide a summary of HEV detection and characterization in farmed ruminants. A total of 1567 papers were retrieved from four search databases that resulted in 35 eligible papers after application of exclusion/inclusion criteria. Studies on HEV in farmed ruminants were mainly based on the detection of HEV RNA and were reported in Africa ( n = 1), America ( n = 3), Asia ( n = 18) and Europe ( n = 13), and focused on a variety of ruminants species, namely cow, goat, sheep, deer, buffalo and yak. The overall pooled prevalence of HEV was 0.02% (0.01-0.03, 95% CI). The subgroup pooled prevalence of HEV RNA was 0.01% (0.00-0.02, 95% CI) in cow milk, stool, serum, liver, intestinal, bile, blood, spleen and rectal swab samples; 0.09% (0.02-0.18, 95% CI) in goat serum, bile, stool, milk, liver, rectal swab and blood samples; 0.01% (0.00-0.04, 95% CI) in sheep stool, serum, milk, blood and liver samples. Most of the HEV genotypes found in farmed ruminants belonged to the zoonotic HEV-3 (subtypes 3a, 3c) and HEV-4 (subtype 4d, 4h), with Rocahepevirus also found. The wide HEV circulation observed in different farmed ruminants raises concerns for the possibility of HEV transmission through products from infected ruminants and alerts for the potential zoonotic route for HEV in ruminant products, such as meat and dairy products. Also, contact exposure to infected farmed animals could be a risk factor. Further research should be conducted in order to understand the circulation of HEV in these animals and its zoonotic potential, as there is currently a lack of data on this topic.

Pigs are a reservoir of hepatitis E virus (HEV), which causes hepatitis in humans. To study the epidemiology of HEV in pig farms, sampling methods are currently used that cause discomfort to pigs, such as rectal sampling. In line with the 3Rs principle, we aimed to evaluate non-invasive methods to detect pens with HEV-shedding pigs. Twenty-eight pens of one farm were sampled cross-sectionally. Individual rectal swabs (IRS) were collected to determine prevalence within pens. Four pen-level samples were compared: a pool of IRS per pen (P), boot socks (BS), oral fluid (OF) and pooled faecal droppings (FD). Each sample was tested by RT-PCR and the sensitivity and specificity of each method was determined by Bayesian latent class analysis. According to IRS, 19/28 pens were HEV positive. BS had a sensitivity of 95% and detected HEV in pens with 10% of pigs shedding; however, specificity was below 30%. FD were comparably accurate to P, with a sensitivity and specificity of 94% and 86%, respectively. BS sampling is thus advised to detect early shedding of HEV or pen contamination, and FD to determine the duration of shedding. This study demonstrates that non-invasive sampling can replace rectal swabs in research on HEV in pigs.

Background: Outbreaks of zoonotic emerging infectious diseases (EIDs) require rapid identification of potential reservoir hosts and mapping disease spread in these hosts to inform risk assessment and adequate control measures. Animals are often understudied when a novel EID is detected in humans and acquisition of animal samples is hampered by practical, ethical, and legal barriers, of which there is currently no clear overview. Therefore, the three aims of this study are (1) to map potentially available collections of animal samples, (2) to assess possibilities and barriers for reuse of these samples and (3) to assess possibilities and barriers for active animal and environmental sampling in the Netherlands., Methods: A literature search was performed to identify ongoing sampling activities and opportunities for reuse or active sampling. Semi-structured interviews with stakeholder organizations were conducted to gain further insight into the three research questions., Results: Various sample collections of surveillance, diagnostic and research activities exist in the Netherlands. Sample size, coverage, storage methods and type of samples collected differs per animal species which influences reuse suitability. Organizations are more likely to share samples, for reuse in outbreak investigations, when they have a pre-existing relationship with the requesting institute. Identified barriers for sharing were, among others, unfamiliarity with legislation and unsuitable data management systems. Active sampling of animals or the environment is possible through several routes. Related barriers are acquiring approval from animal- or property owners, conflicts with anonymization, and time needed to acquire ethical approval., Conclusion: The animal sample collections identified would be very valuable for use in outbreak investigations. Barriers for sharing may be overcome by increasing familiarity with legislation, building (international) sharing networks and agreements before crises occur and developing systems for sample registration and biobanking. Proactive setting up of ethical approvals will allow for rapid animal sample collection to identify EID hosts and potential spillovers., Competing Interests: The authors do not declare any conflicts of interest., (© 2023 Published by Elsevier B.V.)

Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapped by XL-MS since their epitopes overlapped with the epitope recognized by M170. Surprisingly, residues internal in the 146S quaternary structure were also cross-linked to VHH. This probably reflects particle flexibility in solution. Molecular studies of virus-antibody interactions help to further optimize vaccines and improve their quality control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Harmsen, Li, Sun, van der Poel and Dekker.)

Common aims of animal health surveillance systems are the timely detection of emerging diseases and health status monitoring. This study aimed to evaluate the coverage and representativeness of passive surveillance components for cattle and swine in the Netherlands from 2015-2019. The passive surveillance components consisted of a telephone helpdesk for veterinary advice and diagnostic and postmortem facilities. Spatial analysis showed heterogeneity (range in RR = 0.26-5.37) of participation across the Netherlands. Generalized linear mixed models showed that distance to the diagnostic facility and farm density were associated with the number of contacts of farmers with the helpdesk and postmortem examination. The contact rate of veterinary practices was associated with their number of clients, ranging in RR from 0.39 to 1.59. We concluded that the evaluation indicated differences in coverage of the passive surveillance components across regions, farms and veterinary practices. Due to the absence of emerging infections in the study period, we were unable to estimate the consequences of the observed differences for the early detection of disease. Nevertheless, regions and veterinary practices with low participation in passive surveillance might be a risk for early detection, and consequently, further understanding of the motivation to participate in passive surveillance components is needed.

Infection with hepatitis E virus (HEV) is common in both developing and industrialized nations. Genotypes 3 and 4 are increasingly being reported, particularly in high-income countries where the precise extent of HEV transmission via food is currently unclear. Recently, HEV has been found to be excreted in milk; however, data on the potential milk-borne transmission is still lacking or conflicting and warrants further research on the topic. As such, the aim of the present study was to review the current scientific knowledge and to summarize the existing studies in which HEV has been detected in milk. Exhaustive searches were carried out in Mendeley, PubMed, Scopus and Web of Science. A total of 157 papers were retrieved from the four electronic databases. After removing duplicate articles from the databases (n = 30), exclusion criteria identified unrelated research (n = 115). This allowed the identification of 12 eligible papers. To date, studies on HEV detection in milk were mostly from China (n = 5), followed by Egypt (n = 2), Germany (n = 1), Belgium and Holland (n = 1), Turkey (n = 1), Czech Republic (n = 1) and Spain (n = 1) and were focused on a variety of animals (cow, goat, donkey, buffalo, sheep and camel) and humans. Four out of the 12 eligible studies did not find any evidence of HEV in milk. Moreover, 3 out of the 12 studies detected low rates of HEV (0.2-1.8%) and two were based on a low sample size (n = 1 and n = 4). Interestingly, one study showed very high detection rates and also detected HEV genotype 1 in an animal milk sample, an unusual finding since it only occurs in humans, deserving further studies for confirmation and characterization. Two studies detected high prevalence of HEV genotype 4 in bovine samples from China, with one showing indication of the presence of infectious HEV in milk. To date, there is still a small amount of available data on the HEV presence in milk, posing important questions regarding both animal and human health. Thus, further efforts on this potentially underestimated zoonotic route for HEV should be given, warranting further studies on the topic., (© 2022 Wiley-VCH GmbH.)

In the Netherlands, 69 of the 126 (55%) mink farms in total became infected with SARS-CoV-2 in 2020. Despite strict biosecurity measures and extensive epidemiological investigations, the main transmission route remained unclear. A better understanding of SARS-CoV-2 transmission between mink farms is of relevance for countries where mink farming is still common practice and can be used as a case study to improve future emerging disease preparedness. We assessed whether SARS-CoV-2 spilled over from mink to free-ranging animals, and whether free-ranging animals may have played a role in farm-to-farm transmission in the Netherlands. The study encompassed farm visits, farm questionnaires, expert workshops and SARS-CoV-2 RNA and antibody testing of samples from target animal species (bats, birds and free-ranging carnivores). In this study, we show that the open housing system of mink allowed access to birds, bats and most free-ranging carnivores, and that direct and indirect contact with mink was likely after entry, especially for free-ranging carnivores and birds. This allowed SARS-CoV-2 exposure to animals entering the mink farm, and subsequent infection or mechanical carriage by the target animal species. Moreover, mink can escape farms in some cases, and two SARS-CoV-2-positive mink were found outside farm premises. No other SARS-CoV-2-RNA-positive free-ranging animals were detected, suggesting there was no abundant circulation in the species tested during the study period. To investigate previous SARS-CoV-2 infections, SARS-CoV-2 antibody detection using lung extracts of carcasses was set up and validated. One tested beech marten did have SARS-CoV-2 antibodies, but the closest SARS-CoV-2-infected mink farm was outside of its home range, making infection at a mink farm unlikely. Knowing that virus exchange between different species and the formation of animal reservoirs affects SARS-CoV-2 evolution, continued vigilance and monitoring of mink farms and surrounding wildlife remains vital., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

The family Hepeviridae includes enterically transmitted small quasi-enveloped or non-enveloped positive-sense single-stranded RNA viruses infecting mammals and birds (subfamily Orthohepevirinae ) or fish ( Parahepevirinae ). Hepatitis E virus (genus Paslahepevirus ) is responsible for self-limiting acute hepatitis in humans; the infection may become chronic in immunocompromised individuals and extrahepatic manifestations have been described. Avian hepatitis E virus (genus Avihepevirus ) causes hepatitis-splenomegaly syndrome in chickens. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hepeviridae , which is available at www.ictv.global/report/hepeviridae.

Animals like mink, cats and dogs are susceptible to SARS-CoV-2 infection. In the Netherlands, 69 out of 127 mink farms were infected with SARS-CoV-2 between April and November 2020 and all mink on infected farms were culled after SARS-CoV-2 infection to prevent further spread of the virus. On some farms, (feral) cats and dogs were present. This study provides insight into the prevalence of SARS-CoV-2-positive cats and dogs in 10 infected mink farms and their possible role in transmission of the virus. Throat and rectal swabs of 101 cats (12 domestic and 89 feral cats) and 13 dogs of 10 farms were tested for SARS-CoV-2 using PCR. Serological assays were performed on serum samples from 62 adult cats and all 13 dogs. Whole Genome Sequencing was performed on one cat sample. Cat-to-mink transmission parameters were estimated using data from all 10 farms. This study shows evidence of SARS-CoV-2 infection in 12 feral cats and 2 dogs. Eleven cats (18%) and two dogs (15%) tested serologically positive. Three feral cats (3%) and one dog (8%) tested PCR-positive. The sequence generated from the cat throat swab clustered with mink sequences from the same farm. The calculated rate of mink-to-cat transmission showed that cats on average had a chance of 12% (95%CI 10%-18%) of becoming infected by mink, assuming no cat-to-cat transmission. As only feral cats were infected it is most likely that infections in cats were initiated by mink, not by humans. Whether both dogs were infected by mink or humans remains inconclusive. This study presents one of the first reports of interspecies transmission of SARS-CoV-2 that does not involve humans, namely mink-to-cat transmission, which should also be considered as a potential risk for spread of SARS-CoV-2., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

SARS-CoV-2 outbreaks on 69 Dutch mink farms in 2020 were studied to identify risk factors for virus introduction and transmission and to improve surveillance and containment measures. Clinical signs, laboratory test results, and epidemiological aspects were investigated, such as the date and reason of suspicion, housing, farm size and distances, human contact structure, biosecurity measures, and presence of wildlife, pets, pests, and manure management. On seven farms, extensive random sampling was performed, and age, coat color, sex, and clinical signs were recorded. Mild to severe respiratory signs and general diseases such as apathy, reduced feed intake, and increased mortality were detected on 62/69 farms. Throat swabs were more likely to result in virus detection than rectal swabs. Clinical signs differed between virus clusters and were more severe for dark-colored mink, males, and animals infected later during the year. Geographical clustering was found for one virus cluster. Shared personnel could explain some cases, but other transmission routes explaining farm-to-farm spread were not elucidated. An early warning surveillance system, strict biosecurity measures, and a (temporary) ban on mink farming and vaccinating animals and humans can contribute to reducing the risks of the virus spreading and acquisition of potential mutations relevant to human and animal health.

Humans can become infected with hepatitis E virus (HEV) by consumption of undercooked pork. To reduce the burden of HEV in humans, mitigation on pig farms is needed. HEV is found on most pig farms globally, yet within-farm seroprevalence estimates vary considerably. Understanding of the underlying variation in infection dynamics within and between farms currently lacks. Therefore, we investigated HEV infection dynamics by sampling 1711 batches of slaughter pigs from 208 Dutch farms over an 8-month period. Four farm types, conventional, organic, and two types with strict focus on biosecurity, were included. Sera were tested individually with an anti-HEV antibody ELISA and pooled per batch with PCR. All farms delivered seropositive pigs to slaughter, yet batches (resembling farm compartments) had varying results. By combining PCR and ELISA results, infection moment and extent per batch could be classified as low transmission, early, intermediate or late. Cluster analysis of batch infection moments per farm resulted in four clusters with distinct infection patterns. Cluster 1 farms delivered almost exclusively PCR negative, ELISA positive batches to slaughter (PCR - ELISA + ), indicating relatively early age of HEV infection. Cluster 2 and 3 farms delivered 0.3 and 0.7 of batches with intermediate infection moment (PCR + ELISA + ) respectively and only few batches with early infection. Cluster 4 farms delivered low transmission (PCR - ELISA - ) and late infection (PCR + ELISA - ) batches, demonstrating that those farms can prevent or delay HEV transmission to farm compartments. Farm type partly coincided with cluster assignment, indicating that biosecurity and management are related to age of HEV infection., (© 2022. The Author(s).)

Angiotensin converting enzyme 2 (ACE2) is a host cell membrane protein (receptor) that mediates the binding of coronavirus, most notably SARS coronaviruses in the respiratory and gastrointestinal tracts. Although SARS-CoV-2 infection is mainly confined to humans, there have been numerous incidents of spillback (reverse zoonoses) to domestic and captive animals. An absence of information on the spatial distribution of ACE2 in animal tissues limits our understanding of host species susceptibility. Here, we describe the distribution of ACE2 using immunohistochemistry (IHC) on histological sections derived from carnivores, ungulates, primates and chiroptera. Comparison of mink (Neovison vison) and ferret (Mustela putorius furo) respiratory tracts showed substantial differences, demonstrating that ACE2 is present in the lower respiratory tract of mink but not ferrets. The presence of ACE2 in the respiratory tract in some species was much more restricted as indicated by limited immunolabelling in the nasal turbinate, trachea and lungs of cats (Felis catus) and only the nasal turbinate in the golden Syrian hamster (Mesocricetus auratus). In the lungs of other species, ACE2 could be detected on the bronchiolar epithelium of the sheep (Ovis aries), cattle (Bos taurus), European badger (Meles meles), cheetah (Acinonyx jubatus), tiger and lion (Panthera spp.). In addition, ACE2 was present in the nasal mucosa epithelium of the serotine bat (Eptesicus serotinus) but not in pig (Sus scrofa domestica), cattle or sheep. In the intestine, ACE2 immunolabelling was seen on the microvillus of enterocytes (surface of intestine) across various taxa. These results provide anatomical evidence of ACE2 expression in a number of species which will enable further understanding of host susceptibility and tissue tropism of ACE2 receptor-mediated viral infection., (© 2021 Crown copyright. Transboundary and Emerging Diseases published by Wiley-VCH GmbH. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.)

The COVID-19 pandemic has illustrated the need for the development of fast and reliable testing methods for novel, zoonotic, viral diseases in both humans and animals. Pathologies lead to detectable changes in the volatile organic compound (VOC) profile of animals, which can be monitored, thus allowing the development of a rapid VOC-based test. In the current study, we successfully trained honeybees (Apis mellifera) to identify SARS-CoV-2 infected minks (Neovison vison) thanks to Pavlovian conditioning protocols. The bees can be quickly conditioned to respond specifically to infected mink's odours and could therefore be part of a wider SARS-CoV-2 diagnostic system. We tested two different training protocols to evaluate their performance in terms of learning rate, accuracy and memory retention. We designed a non-invasive rapid test in which multiple bees are tested in parallel on the same samples. This provided reliable results regarding a subject's health status. Using the data from the training experiments, we simulated a diagnostic evaluation trial to predict the potential efficacy of our diagnostic test, which yielded a diagnostic sensitivity of 92% and specificity of 86%. We suggest that a honeybee-based diagnostics can offer a reliable and rapid test that provides a readily available, low-input addition to the currently available testing methods. A honeybee-based diagnostic test might be particularly relevant for remote and developing communities that lack the resources and infrastructure required for mainstream testing methods., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The hepatitis E virus (HEV) is responsible for 20 million infections worldwide per year. Although, HEV infection is mostly self-limiting, immunocompromised individuals may evolve toward chronicity. The lack of an efficient small animal model has hampered the study of HEV and the discovery of anti-HEV therapies. Furthermore, new HEV strains, infectious to humans, are being discovered. Human liver-chimeric mice have greatly aided in the understanding of HEV, but only two genotypes (HEV-1 and HEV-3) have been studied in this model. Moreover, the immunodeficient nature of this mouse model does not allow full investigation of the virus and all aspects of its interaction with the host. Recent studies have shown the susceptibility of regular and nude Balb/c mice to a HEV-4 strain (KM01). This model should allow the investigation of the interplay between HEV and the adaptive immune system of its host, and potential immune-mediated complications. Here, we assess the susceptibility of human liver-chimeric and non-humanised mice to a different HEV-4 strain (BeSW67HEV4-2008). We report that humanised mice could be readily infected with this isolate, resulting in an infection pattern comparable to HEV-3 infection. Despite these results and in contrast to KM01, non-humanised mice were not susceptible to infection with this viral strain. Further investigation, using other HEV-4 isolates, is needed to conclusively determine HEV-4 tropism and mouse susceptibility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Collignon, Verhoye, Hakze-Van der Honing, Van der Poel and Meuleman.)

Domestic cats are susceptible to SARS-CoV-2 virus infection and given that they are in close contact with people, assessing the potential risk cats represent for the transmission and maintenance of SARS-CoV-2 is important. Assessing this risk implies quantifying transmission from humans-to-cats, from cats-to-cats and from cats-to-humans. Here we quantified the risk of cat-to-cat transmission by reviewing published literature describing transmission either experimentally or under natural conditions in infected households. Data from these studies were collated to quantify the SARS-CoV-2 reproduction number R 0 among cats. The estimated R 0 was significantly higher than one, hence cats could play a role in the transmission and maintenance of SARS-CoV-2. Questions that remain to be addressed are the risk of transmission from humans-to-cats and cats-to-humans. Further data on household transmission and data on virus levels in both the environment around infected cats and their exhaled air could be a step towards assessing these risks.

Objective: Unprecedented SARS-CoV-2 infections in farmed minks raised immediate concerns regarding transmission to humans and initiated intensive environmental investigations to assess occupational and environmental exposure., Methods: Air sampling was performed at infected Dutch mink farms, at farm premises and at nearby residential sites. A range of other environmental samples were collected from minks' housing units, including bedding materials. SARS-CoV-2 RNA was analysed in all samples by quantitative PCR., Results: Inside the farms, considerable levels of SARS-CoV-2 RNA were found in airborne dust, especially in personal inhalable dust samples (approximately 1000-10 000 copies/m 3 ). Most of the settling dust samples tested positive for SARS-CoV-2 RNA (82%, 75 of 92). SARS-CoV-2 RNA was not detected in outdoor air samples, except for those collected near the entrance of the most recently infected farm. Many samples of minks' housing units and surfaces contained SARS-CoV-2 RNA., Conclusions: Infected mink farms can be highly contaminated with SARS-CoV-2 RNA. This warns of occupational exposure, which was substantiated by considerable SARS-CoV-2 RNA concentrations in personal air samples. Dispersion of SARS-CoV-2 to outdoor air was found to be limited and SARS-CoV-2 RNA was not detected in air samples collected beyond farm premises, implying a negligible risk of environmental exposure to nearby communities. Our occupational and environmental risk assessment is in line with whole genome sequencing analyses showing mink-to-human transmission among farm workers, but no indications of direct zoonotic transmission events to nearby communities., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

In the first wave of the COVID-19 pandemic (April 2020), SARS-CoV-2 was detected in farmed minks and genomic sequencing was performed on mink farms and farm personnel. Here, we describe the outbreak and use sequence data with Bayesian phylodynamic methods to explore SARS-CoV-2 transmission in minks and humans on farms. High number of farm infections (68/126) in minks and farm workers (>50% of farms) were detected, with limited community spread. Three of five initial introductions of SARS-CoV-2 led to subsequent spread between mink farms until November 2020. Viruses belonging to the largest cluster acquired an amino acid substitution in the receptor binding domain of the Spike protein (position 486), evolved faster and spread longer and more widely. Movement of people and distance between farms were statistically significant predictors of virus dispersal between farms. Our study provides novel insights into SARS-CoV-2 transmission between mink farms and highlights the importance of combining genetic information with epidemiological information when investigating outbreaks at the animal-human interface., (© 2021. The Author(s).)

The aim of this pilot study was to determine viral loads and distribution over the total length, at short distances, and in the separate layers of the intestine of virus-infected animals for future inactivation studies. Two calves, two pigs, and two goats were infected with bovine viral diarrhoea virus (BVDV), classical swine fever virus (CSFV), and peste des petits ruminants virus (PPRV), respectively. Homogenously distributed maximum BVDV viral loads were detected in the ileum of both calves, with a mean titer of 6.0 log 10 TCID 50 -eq/g. The viral loads in colon and caecum were not distributed homogenously. In one pig, evenly distributed CSFV mean viral loads of 4.5 and 4.2 log 10 TCID 50 -eq/g were found in the small and large intestines, respectively. Mucosa, submucosa, and muscular layer/serosa showed mean viral loads of 5.3, 3.4, and 4.0 log 10 TCID 50 -eq/g, respectively. Homogenous distribution of PPRV was shown in the ileum of both goats, with a mean viral load of 4.6 log 10 TCID 50 -eq/g. Mean mucosa, submucosa, and muscular layer/serosa viral loads were 3.5, 2.8, and 1.7 log 10 TCID 50 -eq/g, respectively. This pilot study provides essential data for setting up inactivation experiments with intestines derived from experimentally infected animals, in which the level and the homogeneous distribution of intestinal viral loads are required.

In assessing species susceptibility for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and in the search for an appropriate animal model, multiple research groups around the world inoculated a broad range of animal species using various SARS-CoV-2 strains, doses and administration routes. Although in silico analyses based on receptor binding and diverse in vitro cell cultures were valuable, exact prediction of species susceptibility based on these tools proved challenging. Here, we assessed whether precision-cut lung slices (PCLS) could facilitate the selection of animal models, thereby reducing animal experimentation. Pig, hamster and cat PCLS were incubated with SARS-CoV-2 and virus replication was followed over time. Virus replicated efficiently in PCLS from hamsters and cats, while no evidence of replication was obtained for pig PCLS. These data corroborate the findings of many research groups that have investigated the susceptibility of hamsters, pigs and cats towards infection with SARS-CoV-2. Our findings suggest that PCLS can be used as convenient tool for the screening of different animal species for sensitivity to newly emerged viruses. To validate our results obtained in PCLS, we employed the hamster model. Hamsters were inoculated with SARS-CoV-2 via the intranasal route. Susceptibility to infection was evaluated by body weight loss, viral loads in oropharyngeal swabs and respiratory tissues and lung pathology. The broadly used hamster model was further refined by including activity tracking of the hamsters by an activity wheel as a very robust and sensitive parameter for clinical health. In addition, to facilitate the quantification of pathology in the lungs, we devised a semi-quantitative scoring system for evaluating the degree of histological changes in the lungs. The inclusion of these additional parameters refined and enriched the hamster model, allowing for the generation of more data from a single experiment.

Pigs are suspected to be a major source of zoonotic hepatitis E virus (HEV) infection in industrialized countries, but the transmission route(s) from pigs to humans are ill-defined. Sequence comparison of HEV isolates from pigs with those from blood donors and patients in 372 samples collected in the Netherlands in 1998 and 1999 and between 2008 and 2015 showed that all sequences were genotype 3 except for six patients (with travel history). Subgenotype 3c (gt3c) was the most common subtype. While the proportion of gt3c increased significantly between 1998 and 2008, it remained constant between 2008 and 2015. Among the few circulating HEV subtypes, there was no difference observed between the human and the pig isolates. Hepatitis E viruses in humans are very likely to originate from pigs, but it is unclear why HEV gt3c has become the predominant subtype in the Netherlands.

The ongoing coronavirus disease 19s pandemic has yet again demonstrated the importance of the human-animal interface in the emergence of zoonotic diseases, and in particular the role of wildlife and livestock species as potential hosts and virus reservoirs. As most diseases emerge out of the human-animal interface, a better understanding of the specific drivers and mechanisms involved is crucial to prepare for future disease outbreaks. Interactions between wildlife and livestock systems contribute to the emergence of zoonotic diseases, especially in the face of globalization, habitat fragmentation and destruction and climate change. As several groups of viruses and bacteria are more likely to emerge, we focus on pathogenic viruses of the Bunyavirales, Coronaviridae, Flaviviridae, Orthomyxoviridae, and Paramyxoviridae, as well as bacterial species including Mycobacterium sp., Brucella sp., Bacillus anthracis and Coxiella burnetii. Noteworthy, it was difficult to predict the drivers of disease emergence in the past, even for well-known pathogens. Thus, an improved surveillance in hotspot areas and the availability of fast, effective, and adaptable control measures would definitely contribute to preparedness. We here propose strategies to mitigate the risk of emergence and/or re-emergence of prioritized pathogens to prevent future epidemics., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Hepatitis E virus (HEV) genotype 3 and 4 is a zoonosis that causes hepatitis in humans. Humans can become infected by consumption of pork or contact with pigs. Pigs are the main reservoir of the virus worldwide and the virus is present on most pig farms., Main Body: Though HEV is present on most farms, the proportion of infected pigs at slaughter and thus the level of exposure to consumers differs between farms and countries. Understanding the cause of that difference is necessary to install effective measures to lower HEV in pigs at slaughter. Here, HEV studies are reviewed that include infection dynamics of HEV in pigs and on farms, risk factors for HEV farm prevalence, and that describe mechanisms and sources that could generate persistence on farms. Most pigs become infected after maternal immunity has waned, at the end of the nursing or beginning of the fattening phase. Risk factors increasing the likelihood of a high farm prevalence or proportion of actively infected slaughter pigs comprise of factors such as farm demographics, internal and external biosecurity and immunomodulating coinfections. On-farm persistence of HEV is plausible, because of a high transmission rate and a constant influx of susceptible pigs. Environmental sources of HEV that enhance persistence are contaminated manure storages, water and fomites., Conclusion: As HEV is persistently present on most pig farms, current risk mitigation should focus on lowering transmission within farms, especially between farm compartments. Yet, one should be aware of the paradox of increasing the proportion of actively infected pigs at slaughter by reducing transmission insufficiently. Vaccination of pigs may aid HEV control in the future.