Public Health Threat of New, Reemerging, and Neglected Zoonoses in the Industrialized World

Microbiologic infections acquired from animals, known as zoonoses, pose a risk to public health. An estimated 60% of emerging human pathogens are zoonotic. Of these pathogens, >71% have wildlife origins. These pathogens can switch hosts by acquiring new genetic combinations that have altered pathogenic potential or by changes in behavior or socioeconomic, environmental, or ecologic characteristics of the hosts. We discuss causal factors that influence the dynamics associated with emergence or reemergence of zoonoses, particularly in the industrialized world, and highlight selected examples to provide a comprehensive view of their range and diversity

European Bat Lyssaviruses, the Netherlands

Genotype 5 lyssaviruses are endemic in the Netherlands, and can cause fatal infections in humans

Sentinel chicken surveillance reveals previously undetected circulation of West Nile virus in the Netherlands

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.</p

Survival and Elimination of Hepatitis E Virus : A Review

Recently, foodborne transmission of hepatitis E virus (HEV) has become a concern due to the identification of undercooked pork products as a risk factor for infection. The limited number of studies which have been performed indicate that HEV could remain infectious at temperatures used in some cooking regimes, although inactivation by heating at 71 °C for 20 min has been demonstrated. There are significant gaps in our knowledge regarding the survival of HEV in foods and the environment (including food contact surfaces), and also regarding the effect of elimination procedures used in food supply chain settings. The lack of a reliable infectivity assay has hampered extensive study. It is recommended that studies be undertaken to develop an efficient propagation system (based on in vitro cell culture), to facilitate the acquisition of extensive information on the survival of HEV in food and the environment, and its response to disinfection and elimination procedures.</p

Study of Hepatitis E Virus-4 Infection in Human Liver-Chimeric, Immunodeficient, and Immunocompetent Mice

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

Hepatitis E Virus in Farmed Rabbits, Wild Rabbits and Petting Farm Rabbits in the Netherlands

Rabbits have been suggested as a zoonotic source of Hepatitis E virus. Phylogenetic analysis of HEV isolates from farmed, wild and pet rabbits in the Netherlands (23, 0, and 60 % respectively) showed them to be grouped amongst published rabbit HEV sequences and distinct from most human isolates. Dutch rabbits are unlikely to be a zoonotic source.</p

Mapping of foot-and-mouth disease virus antigenic sites recognized by single-domain antibodies reveals different 146S particle specific sites and particle flexibility

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

Development and validation of SYBR Green- and probe-based reverse-transcription real-time PCR assays for detection of the S and M segments of Schmallenberg virus

Schmallenberg virus (SBV), discovered in Germany in 2011, causes congenital malformations in ruminants. Reverse-transcription real-time PCR (RT-rtPCR) assays based on various segments of SBV have been developed for molecular detection. We developed alternative RT-rtPCR assays for SBV detection to avoid earlier reported mutations and hypervariable regions of the S and M segments of the viral genome. For SYBR Green-based detection of the S segment, the R2 value and efficiency of the developed assay were 0.99 and 99%, respectively. For probe-based S segment detection, 2 assays were developed; the first had an R2 value of 0.99 and 102% efficiency, and the second had a R2 value of 0.98 and 86% efficiency. The probe-based M segment assay had an R2 value of 1.00 and 103% efficiency. Detection limits of the RT-rtPCR assays with new primer sets were 102 and 101 copies/µL for the S and M segments, respectively. Field samples from cattle and sheep were also used for primary validation of the developed assays. Our assays should be suitable for SBV detection in ruminants and for in vitro studies of various SBV strains.</p

Salt inactivation of classical swine fever virus and African swine fever virus in porcine intestines confirms the existing in vitro casings model

Natural casings, to be used as sausage containers, are being traded worldwide and may be contaminated with contagious viruses. Standard processing of such natural casings is by salt treatment with a duration of 30 days before shipment. Since information is lacking about the efficacy of these virus inactivation procedures, an in vitro 3D collagen matrix model, mimicking natural casings, was developed previously to determine the efficacy of salt to inactivate specific viruses. To validate this model, a comparison in vivo experiment was performed using intestines of pigs experimentally infected with African swine fever virus (ASFV) and classical swine fever virus (CSFV). Decimal reduction (D) values, were determined at 4 °C, 12 °C, 20 °C and 25 °C. The standard salt processing procedure showed an efficient inactivation of ASFV and CSFV over time in a temperature dependent way. Dintestine values of both viruses, treated with the standard salt treatment, were in line with the Dcollagen values. It was concluded that these results underline the suitability of the 3D collagen matrix model to determine virus inactivation and to replace animal experiments. Furthermore, an increase in storage time for standard salt processed casings derived from CSFV endemic regions is highly recommended for an efficient inactivation of CSFV.</p