S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019. Researchers have identified close relatives to SARS-CoV-2 in bats (1) and pangolins (order Pholidota) (2,3). Whether the pandemic was initiated by direct transmission from bats or through an intermediate mammalian host is still under debate (4). During the 2002-2004 severe acute respiratory syndrome pandemic, researchers documented the causative virus in raccoon dogs (Nyctereutes procyonoides) in China, indicating that these animals might have been intermediate hosts for the virus (5). Fur producers in China own >14 million captive raccoon dogs, accounting for ≈99% of the global share of raccoon dogs (6) (Appendix Figure 1, panel A, https:// wwwnc.cdc.gov/EID/article/26/12/20-3733-App1. pdf). However, whether these animals are susceptible to SARS-CoV-2 is unknown. Using our established study design (7), we characterized susceptibility, viral shedding, transmission potential, serologic reactions, and pathologic lesions of raccoon dogs after experimental SARS-CoV-2 infection.

Orthobunyaviruses (OBVs) form a distinct genus of arthropod-borne bunyaviruses that can cause severe disease upon zoonotic transmission to humans. Antigenic drift or genome segment re-assortment have in the past resulted in new pathogenic OBVs, making them potential candidates for causing emerging zoonoses in the future. Low-resolution electron cryo-tomography studies have shown that OBV particles feature prominent trimeric spikes, but their molecular organization remained unknown. Here we report X-ray crystallography studies of four different OBVs showing that the spikes are formed by an N-terminal extension of the fusion glycoprotein Gc. Using Schmallenberg virus, a recently emerged OBV, we also show that the projecting spike is the major target of the neutralizing antibody response, and provide X-ray structures in complex with two protecting antibodies. We further show that immunization of mice with the spike domains elicits virtually sterilizing immunity, providing fundamental knowledge essential in the preparation for potential newly emerging OBV zoonoses.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe adverse effect of ChAdOx1 nCoV-19 COVID-19 vaccine (Vaxzevria) and COVID-19 vaccine Janssen (Ad26.COV2.S), and associated with unusual thrombosis. VITT is caused by anti-platelet factor 4 (PF4) antibodies activating platelets through their FcgRIIa receptors. Antibodies activating platelets through FcgRIIa receptors have also been identified in COVID-19 patients. These findings raise concern that vaccination-induced antibodies against anti-SARS-CoV-2 spike protein cause thrombosis by cross-reacting with PF4. Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared using in-silico prediction tools and 3D-modelling. The SARS-CoV-2 spike protein and PF4 share at least one similar epitope. Reactivity of purified anti-PF4 antibodies from patients with VITT was tested against recombinant SARS-CoV-2 spike protein. However, none of the affinity-purified anti-PF4 antibodies from 14 VITT patients cross-reacted with SARS-CoV-2 spike protein. Sera from 222 PCR-confirmed COVID-19 patients from five European centers were tested by PF4/heparin ELISA and PF4-dependent platelet activation assays. We found anti-PF4 antibodies in 19 of 222 (8.6%) COVID-19 patient sera. However, only four showed weak to moderate platelet activation in the presence of PF4, and none of these patients developed thrombotic complications. Among 10 of 222 (4.5%) COVID-19 patients with thrombosis, none showed PF4-dependent platelet-activating antibodies. In conclusion, antibodies against PF4 induced by vaccination do not cross-react with the SARS-CoV-2 spike protein, indicating that the intended vaccine-induced immune response against SARS-CoV-2 spike protein is not the trigger of VITT. PF4-reactive antibodies found in COVID-19 patients of the present study were not associated with thrombotic complications.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused a pandemic with millions of infected humans and hundreds of thousands of fatalities. As the novel disease – referred to as COVID‐19 – unfolded, occasional anthropozoonotic infections of animals by owners or caretakers were reported in dogs, felid species and farmed mink. Further species were shown to be susceptible under experimental conditions. The extent of natural infections of animals, however, is still largely unknown. Serological methods will be useful tools for tracing SARS‐CoV‐2 infections in animals once test systems are evaluated for use in different species. Here, we developed an indirect multi‐species ELISA based on the receptor‐binding domain (RBD) of SARS‐CoV‐2. The newly established ELISA was evaluated using 59 sera of infected or vaccinated animals, including ferrets, raccoon dogs, hamsters, rabbits, chickens, cattle and a cat, and a total of 220 antibody‐negative sera of the same animal species. Overall, a diagnostic specificity of 100.0% and sensitivity of 98.31% were achieved, and the functionality with every species included in this study could be demonstrated. Hence, a versatile and reliable ELISA protocol was established that enables high‐throughput antibody detection in a broad range of animal species, which may be used for outbreak investigations, to assess the seroprevalence in susceptible species or to screen for reservoir or intermediate hosts.

Schmallenberg virus (SBV) is transmitted by insect vectors, and therefore vaccination is one of the most important tools of disease control. In our study, novel subunit vaccines on the basis of an amino-terminal domain of SBV Gc of 234 amino acids (“Gc Amino”) first were tested and selected using a lethal small animal challenge model and then the best performing formulations also were tested in cattle. We could show that neither E. coli expressed nor the reduced form of “Gc Amino” protected from SBV infection. In contrast, both, immunization with “Gc Amino”-encoding DNA plasmids and “Gc-amino” expressed in a mammalian system, conferred protection in up to 66% of the animals. Interestingly, the best performance was achieved with a multivalent antigen containing the covalently linked Gc domains of both, SBV and the related Akabane virus. All vaccinated cattle and mice were fully protected against SBV challenge infection. Furthermore, in the absence of antibodies against the viral N-protein, differentiation between vaccinated and field-infected animals allows an SBV marker vaccination concept. Moreover, the presented vaccine design also could be tested for other members of the Simbu serogroup and might allow the inclusion of additional immunogenic domains.