SARS-CoV-2 Infection and Clinical Signs in Cats and Dogs from Confirmed Positive Households in Germany.

On a global scale, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to the health of the human population. Not only humans can be infected, but also their companion animals. The antibody status of 115 cats and 170 dogs, originating from 177 German households known to have been SARS-CoV-2 positive, was determined by enzyme-linked immunosorbent assay (ELISA), and the results were combined with information gathered from a questionnaire that was completed by the owner(s) of the animals. The true seroprevalences of SARS-CoV-2 among cats and dogs were 42.5% (95% CI 33.5-51.9) and 56.8% (95% CI 49.1-64.4), respectively. In a multivariable logistic regression accounting for data clustered in households, for cats, the number of infected humans in the household and an above-average contact intensity turned out to be significant risk factors; contact with humans outside the household was a protective factor. For dogs, on the contrary, contact outside the household was a risk factor, and reduced contact, once the human infection was known, was a significant protective factor. No significant association was found between reported clinical signs in animals and their antibody status, and no spatial clustering of positive test results was identified.

No indication for SARS-CoV-2 transmission to pet ferrets, in five cities in Poland, 2021 - antibody testing among ferrets living with owners infected with SARS-CoV-2 or free of infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in China by the end of 2019 and was responsible for a pandemic in the human population that resulted in millions of deaths worldwide. Since the beginning of the pandemic, the role of animals as spill-over or reservoir hosts was discussed. In addition to cats and dogs, ferrets are becoming increasingly popular as companion animals. Under experimental conditions, ferrets are susceptible to SARS-CoV-2 and it appears that they can also be infected through contact with a SARS-CoV-2 positive owner. However, there is still little information available regarding these natural infections. Here, we serologically tested samples collected from pet ferrets (n = 45) from Poland between June and September 2021. Of the ferrets that were included in the study, 29% (13/45) had contact with owners with confirmed SARS-CoV-2 infections. Nevertheless, SARS-CoV-2-specific antibodies could not be detected in any of the animals, independent of the infection status of the owner. The obtained results suggest that ferrets cannot be readily infected with SARS-CoV-2 under natural conditions, even after prolonged contact with infected humans. However, due to the rapid mutation rate of this virus, it is important to include ferrets in future monitoring studies.

Efficacy of an unmodified bivalent mRNA vaccine against SARS-CoV-2 variants in female small animal models.

Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve control of the COVID-19 pandemic. We compare monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein in a transgenic mouse and a Wistar rat model. The blended low-dose bivalent mRNA vaccine contains half the mRNA of each respective monovalent vaccine, but induces comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, antigen-specific CD4+ and CD8+ responses, and protects transgenic female mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduces viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized female Wistar rats also contain neutralizing antibodies against the B.1.1.529 (Omicron BA.1 and BA.5) variants. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins are feasible approaches for extending the coverage of vaccines for emerging and co-circulating SARS-CoV-2 variants.

No Evidence for the Presence of SARS-CoV-2 in Bank Voles and Other Rodents in Germany, 2020-2022.

Rodentia is the most speciose mammalian order, found across the globe, with some species occurring in close proximity to humans. Furthermore, rodents are known hosts for a variety of zoonotic pathogens. Among other animal species, rodents came into focus when the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spread through human populations across the globe, initially as laboratory animals to study the viral pathogenesis and to test countermeasures. Under experimental conditions, some rodent species including several cricetid species are susceptible to SARS-CoV-2 infection and a few of them can transmit the virus to conspecifics. To investigate whether SARS-CoV-2 is also spreading in wild rodent populations in Germany, we serologically tested samples of free-ranging bank voles (Myodes glareolus, n = 694), common voles (Microtus arvalis, n = 2), house mice (Mus musculus, n = 27), brown or Norway rats (Rattus norvegicus, n = 97) and Apodemus species (n = 8) for antibodies against the virus. The samples were collected from 2020 to 2022 in seven German federal states. All but one sample tested negative by a multispecies ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2. The remaining sample, from a common vole collected in 2021, was within the inconclusive range of the RBD-ELISA, but this result could not be confirmed by a surrogate virus neutralization test as the sample gave a negative result in this test. These results indicate that SARS-CoV-2 has not become highly prevalent in wild rodent populations in Germany.

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype.

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.

High Seroprevalence against SARS-CoV-2 among Dogs and Cats, Poland, 2021/2022.

The coronavirus SARS-CoV-2 is responsible for a pandemic in the human population that has unfolded since the beginning of 2020 and has led to millions of deaths globally. Apart from humans, SARS-CoV-2 has been confirmed in various animal species, including felines, canines, mustelids, and primates. Of these species, dogs and cats are the most popular companion animals worldwide. Several seroprevalence studies have already been performed in these animal species; however, the results vary depending on the location and especially the time of sampling. Here, serum samples were collected from a total of 388 dogs and 243 cats from three veterinary clinics in two cities (Gdansk and Olsztyn) in Poland between October 2021 and February 2022, when the country was in the midst of the fourth wave of viral spread. All sera were tested for antibodies against SARS-CoV-2 by a multispecies ELISA based on the receptor-binding domain and by an indirect immunofluorescence assay (iIFA). Overall, 18.9% of the feline sera and 16.0% of the canine sera tested positive using ELISA and iIFA. This relatively high seroprevalence among randomly selected animals is most likely related to the high case numbers in the human population and indicates a continuous occurrence of transspecies virus transmissions from infected owners to their pets. Hence, dogs and cats should be included in monitoring studies and/or outbreak investigations for a better understanding of the epidemiology of this virus.

Antibodies against SARS-CoV-2 Suggestive of Single Events of Spillover to Cattle, Germany.

Human infection with SARS-CoV-2 poses a risk for transmission to animals. To characterize the risk for cattle, we serologically investigated 1,000 samples collected from cattle in Germany in late 2021. Eleven antibody-positive samples indicated that cattle may be occasionally infected by contact with SARS-CoV-2-positive keepers, but we found no indication of further spread.

Serological screening in wild ruminants in Germany, 2021/2022: No evidence of SARS-CoV-2, bluetongue virus or pestivirus spread but high seroprevalences against Schmallenberg virus.

Wildlife animals may be susceptible to multiple infectious agents of public health or veterinary relevance, thereby potentially forming a reservoir that bears the constant risk of re-introduction into the human or livestock population. Here, we serologically investigated 493 wild ruminant samples collected in the 2021/2022 hunting season in Germany for the presence of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four viruses pathogenic to domestic ruminants, namely, the orthobunyavirus Schmallenberg virus (SBV), the reovirus bluetongue virus (BTV) and ruminant pestiviruses like bovine viral diarrhoea virus or border disease virus. The animal species comprised fallow deer, red deer, roe deer, mouflon and wisent. For coronavirus serology, additional 307 fallow, roe and red deer samples collected between 2017 and 2020 at three military training areas were included. While antibodies against SBV could be detected in about 13.6% of the samples collected in 2021/2022, only one fallow deer of unknown age tested positive for anti-BTV antibodies, and all samples reacted negative for antibodies against ruminant pestiviruses. In an ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2, 25 out of 493 (5.1%) samples collected in autumn and winter 2021/2022 scored positive. This sero-reactivity could not be confirmed by the highly specific virus neutralisation test, occurred also in 2017, 2018 and 2019, that is, prior to the human SARS-CoV-2 pandemic, and was likewise observed against the RBD of the related SARS-CoV-1. Therefore, the SARS-CoV-2 sero-reactivity was most likely induced by another hitherto unknown deer virus belonging to the subgenus Sarbecovirus of betacoronaviruses.

Prevalence of anti-severe acute respiratory syndrome coronavirus 2 antibodies in cats in Germany and other European countries in the early phase of the coronavirus disease-19 pandemic.

During the first months of the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cases of human-to-cat transmission were reported. Seroconversion was shown in cats infected under experimental and natural conditions. This large-scale survey of 1,005 serum samples was conducted to investigate anti-SARS-CoV-2 antibody prevalence in domestic cats during the first 7 months of the pandemic in Germany and other European countries. In addition, we compared the sensitivity and specificity of two multispecies SARS-CoV-2 antibody enzyme-linked immunosorbent assays (ELISA). Results were confirmed by using an indirect immunofluorescence test (iIFT) and a surrogate virus neutralization test (sVNT). Sera that were highly positive for feline coronavirus (FCoV) antibodies (n = 103) were included to correct for cross-reactivity of the tests used. Our results showed an overall SARS-CoV-2 seropositivity of 1.9% (n = 19) in a receptor-binding domain (RBD)-based ELISA, additional 0.8% (n = 8) were giving inconclusive results. In contrast, a nucleocapsid-based ELISA revealed 0.5% (n = 5) positive and 0.2% (n = 2) inconclusive results. While the iIFT and sVNT confirmed 100% of positive and 50%-57.1% of the doubtful results as determined in the RBD ELISA, the nucleocapsid-based assay showed a high discrepancy and only one of the five positive results could be confirmed. The results indicate significant deficits of the nucleocapsid-based ELISA with respect to sensitivity and specificity. Due to a significantly higher rate (5.8%) of positive results in the group of highly FCoV antibody-positive samples, cross-reactivity of the FCoV-ELISA with SARS-CoV-2 antibodies cannot be excluded. Furthermore, we investigated the impact of direct contact of domestic cats (n = 23) to SARS-CoV-2 positive owners. Considering one inconclusive result, which got confirmed by iIFT, this exposure did not lead to a significantly higher prevalence (4.4%; p = .358) among tested subjects. Overall, we conclude that cats are a negligible entity with respect to virus transmission in Europe.

Saving Resources: SARS-CoV-2 Diagnostics by Real-Time RT-PCR Using Reduced Reaction Volumes.

Since the beginning of 2020, the betacoronavirus SARS-CoV-2 is causing a global pandemic of an acute respiratory disease termed COVID-19. The diagnostics of the novel disease is primarily based on direct virus detection by RT-PCR; however, the availability of test kits may become a major bottleneck, when millions of tests are performed per week. To increase the flexibility of SARS-CoV-2 diagnostics, three real-time RT-PCR assays listed on the homepage of the World Health Organization were selected and investigated regarding their compatibility with three different RT-PCR kits. Furthermore, the reaction volume of the PCR chemistry was reduced up to half of the original protocol to make the individual reactions more cost- and resource-effective. When testing dilution series of culture-grown virus, nearly identical quantification cycle values (Cq) were obtained for all RT-PCR assay/chemistry combinations. Regarding the SARS-CoV-2 detection in clinical samples, agreeing results were obtained for all combinations for virus negative specimens and swabs containing high to medium viral genome loads. In cases of very low SARS-CoV-2 genome loads (Cq > 36), inconsistent results were observed, with some test runs scoring negative and some positive. However, no preference of a specific target within the viral genome (E, RdRp, or N) or of a certain chemistry was seen. In summary, a reduction of the reaction volume and the type of PCR chemistry did not influence the PCR sensitivity.

Detection of SARS-CoV-2 variant B.1.1.7 in a cat in Germany.

Several non-variant of concern SARS-CoV-2 infections in pets have been reported as documented in the OIE and GISAID databases and there is only one fully documented case of an alpha variant of concern (VOC)(B.1.1.7) in the United States so far. Here, we describe the first case in a cat infected with the alpha SARS-CoV-2 variant in Germany. A cat suffering from pneumonia was presented to a veterinary practice. The pneumonia was treated symptomatically, but 16 days later the cat was presented again. Since the owner had been tested positive for a SARS-CoV-2 infection in the meantime, swab samples were taken from the cat and analyzed for SARS-CoV-2 specific nucleic acids. The various RT-qPCR analyses and whole-genome sequencing revealed the presence of the SARS-CoV-2 B.1.1.7 variant in this cat. This study shows that pets living in close contact with SARS-CoV-2 B.1.1.7 infected owners can contract this virus and also suffer from a respiratory disease. It is not clear yet whether onward transmissions to other cats and humans can occur. To minimize transmission risks, pet owners and veterinarians should comply to the hygienic rules published by OIE and others. It must be stated, that infections of cats with SARS-CoV-2 is still a rare event. Cats with clinical signs of a respiratory disease should be presented to a veterinarian, who will decide on further steps.

Serological Detection of SARS-CoV-2 Antibodies in Naturally-Infected Mink and Other Experimentally-Infected Animals.

The recent emergence of SARS-CoV-2 in humans from a yet unidentified animal reservoir and the capacity of the virus to naturally infect pets, farmed animals and potentially wild animals has highlighted the need for serological surveillance tools. In this study, the luciferase immunoprecipitation systems (LIPS), employing the spike (S) and nucleocapsid proteins (N) of SARS-CoV-2, was used to examine the suitability of the assay for antibody detection in different animal species. Sera from SARS-CoV-2 naturally-infected mink (n = 77), SARS-CoV-2 experimentally-infected ferrets, fruit bats and hamsters and a rabbit vaccinated with a purified spike protein were examined for antibodies using the SARS-CoV-2 N and/or S proteins. From comparison with the known neutralization status of the serum samples, statistical analyses including calculation of the Spearman rank-order-correlation coefficient and Cohen's kappa agreement were used to interpret the antibody results and diagnostic performance. The LIPS immunoassay robustly detected the presence of viral antibodies in naturally infected SARS-CoV-2 mink, experimentally infected ferrets, fruit bats and hamsters as well as in an immunized rabbit. For the SARS-CoV-2-LIPS-S assay, there was a good level of discrimination between the positive and negative samples for each of the five species tested with 100% agreement with the virus neutralization results. In contrast, the SARS-CoV-2-LIPS-N assay did not consistently differentiate between SARS-CoV-2 positive and negative sera. This study demonstrates the suitability of the SARS-CoV-2-LIPS-S assay for the sero-surveillance of SARS-CoV-2 infection in a range of animal species.

Evaluation of a multi-species SARS-CoV-2 surrogate virus neutralization test.

Assays to measure SARS-CoV-2-specific neutralizing antibodies are important to monitor seroprevalence, to study asymptomatic infections and to reveal (intermediate) hosts. A recently developed assay, the surrogate virus-neutralization test (sVNT) is a quick and commercially available alternative to the "gold standard" virus neutralization assay using authentic virus, and does not require processing at BSL-3 level. The assay relies on the inhibition of binding of the receptor binding domain (RBD) on the spike (S) protein to human angiotensin-converting enzyme 2 (hACE2) by antibodies present in sera. As the sVNT does not require species- or isotype-specific conjugates, it can be similarly used for antibody detection in human and animal sera. In this study, we used 298 sera from PCR-confirmed COVID-19 patients and 151 sera from patients confirmed with other coronavirus or other (respiratory) infections, to evaluate the performance of the sVNT. To analyze the use of the assay in a One Health setting, we studied the presence of RBD-binding antibodies in 154 sera from nine animal species (cynomolgus and rhesus macaques, ferrets, rabbits, hamsters, cats, cattle, mink and dromedary camels). The sVNT showed a moderate to high sensitivity and a high specificity using sera from confirmed COVID-19 patients (91.3% and 100%, respectively) and animal sera (93.9% and 100%), however it lacked sensitivity to detect low titers. Significant correlations were found between the sVNT outcomes and PRNT50 and the Wantai total Ig and IgM ELISAs. While species-specific validation will be essential, our results show that the sVNT holds promise in detecting RBD-binding antibodies in multiple species.

CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2.

The ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies.

Development of a Modular Vaccine Platform for Multimeric Antigen Display Using an Orthobunyavirus Model.

Emerging infectious diseases represent an increasing threat to human and animal health. Therefore, safe and effective vaccines that could be available within a short time frame after an outbreak are required for adequate prevention and control. Here, we developed a robust and versatile self-assembling multimeric protein scaffold particle (MPSP) vaccine platform using lumazine synthase (LS) from Aquifex aeolicus. This scaffold allowed the presentation of peptide epitopes by genetic fusion as well as the presentation of large antigens by bacterial superglue-based conjugation to the pre-assembled particle. Using the orthobunyavirus model Schmallenberg virus (SBV) we designed MPSPs presenting major immunogens of SBV and assessed their efficacy in a mouse model as well as in cattle, a target species of SBV. All prototype vaccines conferred protection from viral challenge infection and the multivalent presentation of the selected antigens on the MPSP markedly improved their immunogenicity compared to the monomeric subunits. Even a single shot vaccination protected about 80% of mice from an otherwise lethal dose of SBV. Most importantly, the MPSPs induced a virtually sterile immunity in cattle. Altogether, LS represents a promising platform for modular and rapid vaccine design.

Prolonged SARS-CoV-2 RNA Shedding from Therapy Cat after Cluster Outbreak in Retirement Home.

We report a therapy cat in a nursing home in Germany infected with severe acute respiratory syndrome coronavirus 2 during a cluster outbreak in the home residents. Although we confirmed prolonged presence of virus RNA in the asymptomatic cat, genome sequencing showed no further role of the cat in human infections on site.

The Second Wave of SARS-CoV-2 Circulation-Antibody Detection in the Domestic Cat Population in Germany.

Registered cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in the German human population increased rapidly during the second wave of the SARS-CoV-2 pandemic in winter 2020/21. Since domestic cats are susceptible to SARS-CoV-2, the occurrence of trans-species transmission needs to be monitored. A previous serosurvey during the first wave of the pandemic detected antibodies against SARS-CoV-2 in 0.65% of feline serum samples that were randomly sampled across Germany. In the here-presented follow-up study that was conducted from September 2020 to February 2021, the seroprevalence rose to 1.36% (16/1173). This doubling of the seroprevalence in cats is in line with the rise of reported cases in the human population and indicates a continuous occurrence of trans-species transmission from infected owners to their cats.

SARS-CoV-2 in animals: From potential hosts to animal models.

Within only one year after the first detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), nearly 100 million infections were reported in the human population globally, with more than two million fatal cases. While SARS-CoV-2 most likely originated from a natural wildlife reservoir, neither the immediate viral precursor nor the reservoir or intermediate hosts have been identified conclusively. Due to its zoonotic origin, SARS-CoV-2 may also be relevant to animals. Thus, to evaluate the host range of the virus and to assess the risk to act as potential animal reservoir, a large number of different animal species were experimentally infected with SARS-CoV-2 or monitored in the field in the last months. In this review, we provide an update on studies describing permissive and resistant animal species. Using a scoring system based on viral genome detection subsequent to SARS-CoV-2 inoculation, seroconversion, the development of clinical signs and transmission to conspecifics or humans, the susceptibility of diverse animal species was classified on a semi-quantitative scale. While major livestock species such as pigs, cattle and poultry are mostly resistant, companion animals appear moderately susceptible, while several model animal species used in research, including several Cricetidae species and non-human primates, are highly susceptible to SARS-CoV-2 infection. By natural infections, it became obvious that American minks (Neovison vison) in fur farms, e.g., in the Netherlands and Denmark are highly susceptible resulting in local epidemics in these animals.

Experimental SARS-CoV-2 Infection of Bank Voles.

After experimental inoculation, severe acute respiratory syndrome coronavirus 2 infection was confirmed in bank voles by seroconversion within 8 days and detection of viral RNA in nasal tissue for up to 21 days. However, transmission to contact animals was not detected. Thus, bank voles are unlikely to establish effective transmission cycles in nature.

Occurrence of Antibodies against SARS-CoV-2 in the Domestic Cat Population of Germany.

Domestic cats (Felis catus) are popular companion animals that live in close contact with their human owners. Therefore, the risk of a trans-species spreading event between domestic cats and humans is ever-present. Shortly after the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid spread around the world, the role of domestic cats in the transmission cycle was questioned. In the present study, the first large-scale survey of antibody occurrence in the domestic cat population in Germany was conducted, in order to assess the incidence of naturally occurring human to cat transmission of SARS-CoV-2. A total of 920 serum samples, which were collected from April to September of 2020, were screened by an indirect multispecies ELISA. Positive samples were verified using an indirect immunofluorescence test (iIFT) and additionally tested for neutralizing antibodies. Furthermore, serum samples were screened for antibodies against feline coronavirus (FCoV), in order to rule out cross-reactivity in the described test systems. Overall, 0.69% (6/920) of serum samples were found to be positive for antibodies against SARS-CoV-2 by ELISA and iIFT. Two of these reactive sera also displayed neutralizing antibodies. No cross-reactivity with FCoV-specific antibodies was observed. The finding of SARS-CoV-2 antibody-positive serum samples in the domestic cat population of Germany, during a period when the incidence of human infection in the country was still rather low, indicates that human-to-cat transmission of SARS-CoV-2 happens, but there is no indication of SARS-CoV-2 circulation in cats.