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.

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.

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.

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.

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.

Long-term presence of tick-borne encephalitis virus in experimentally infected bank voles (Myodes glareolus).

Tick-borne encephalitis virus (TBEV) is a vector-borne pathogen that can cause serious neurological symptoms in humans. Across large parts of Eurasia TBEV is found in three traditional subtypes: the European, the Siberian and the Far-eastern subtype. Small mammalian animals play an important role in the transmission cycle as they enable the spread of TBEV among the vector tick population. To assess the impact of TBEV infection on its natural hosts, outbred bank voles (Myodes glareolus) were inoculated with one out of four European TBEV strains. Three of these TBEV strains were recently isolated in Germany. The forth one was the TBEV reference strain Neudörfl. Sampling points at 7, 14, 28, and 56 days post inoculation allowed the characterization of the course of infection. At each time point, six animals per strain were euthanized and eleven organ samples (brain, spine, lung, heart, small and large intestine, liver, spleen, kidney, bladder, sexual organ) as well as whole blood and serum samples were collected. The majority of bank voles (92/96) remained clinically unaffected after the inoculation with TBEV, but still developed a systemic infection during the first week, which transitioned to a viraemia and an infestation of the brain in some animals for the remainder of the first month. Viral RNA was found in whole blood samples of several animals (50/96), but only in a small fraction of the corresponding serum samples (4/50). From the whole blood, virus was successfully reisolated in cell culture until 14 days after inoculation. Less than five percent of all inoculated bank voles (4/96) displayed signs of distress in combination with a rapid weight loss and had to be euthanized prematurely. Overall, the recently isolated TBEV strains showed marked differences, such as a more frequent development of long-term viraemia and a higher detection rate of viral RNA in various organs, in comparison to the reference strain Neudörfl. Overall, our data suggest that the bank vole is a potential amplifying host in the TBEV transmission cycle and appears to be highly adapted to circulating TBEV strains.

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.

No evidence for African swine fever virus DNA in haematophagous arthropods collected at wild boar baiting sites in Estonia.

African swine fever (ASF) is a highly pathogenic viral disease affecting all Suidae, with Ornithodoros moubata complex soft ticks acting as the biological arthropod vectors of the causative agent, African swine fever virus (ASFV). While ASFV is also transmissible via direct contact, pig products and fomites, other arthropods may be involved in virus transmission and persistence. Therefore, we checked various groups of blood-feeding arthropods collected during summer 2017 in wild boar habitats on the Estonian Island of Saaremaa for the presence of ASFV. Saaremaa had the highest ASF infection prevalences in Estonia in 2017, with an incidence of 9% among hunted wild boar. In addition to ASFV, we tested for other selected pathogens. In total, 784 ticks, 6,274 culicoid biting midges, 77 tabanids and 757 mosquitoes were tested as individuals or pools. No ASFV-DNA was found in any of them although about 20% of the tick samples tested positive for swine DNA. By contrast, tick-borne encephalitis virus-RNA was detected in one out of 37 tick pools (2.7%) and Borrelia burgdorferi s.l.-DNA in 20 individual ticks and 17 tick pools (25.2% of all samples). No Schmallenberg virus was detected in the Culicoides specimens. In conclusion, we found no evidence for Ixodes ricinus ticks, Culicoides punctatus and Obsoletus complex biting midges, Aedes spp., Anopheles spp. and Culiseta annulata mosquitoes, and Haematopota pluvialis tabanids playing a role in ASFV transmission in the wild boar population in Estonia.

Multi-species ELISA for the detection of antibodies against SARS-CoV-2 in animals.

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.

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.

Seroprevalences of Newly Discovered Porcine Pestiviruses in German Pig Farms.

Several novel porcine pestiviruses that are linked to disease outbreaks in commercial pig farms were discovered during recent years. Bungowannah pestivirus (BuPV; new species Pestivirus F) causes sudden death in young pigs, but has only ever been isolated in the Australian region Bungowannah. Atypical porcine pestivirus (APPV; new species Pestivirus K) on the other hand has been found in multiple countries worldwide and is potentially linked to congenital tremor, a disease that causes considerable production problems in pig farms. To assess the seroprevalences of both viruses in German commercial farms during the years 2009/10 and 2018, two approaches were selected. Antibodies against Pestivirus F were detected by a traditional in-house indirect immunofluorescence test against the culture-grown virus isolate, while for the detection of Pestivirus K-specific antibodies, a newly developed test system utilizing a chimeric construct of bovine viral diarrhea virus 1 (BVDV-1; species Pestivirus A) containing the E1 and E2 encoding sequences of APPV was established. A total of 1115 samples originating from 122 farms located in seven German federal states were investigated. Antibodies against Bungowannah virus could not be detected, confirming the absence of this virus in other regions than the initially affected Australian pig farm complex. In contrast, antibodies against APPV were highly prevalent throughout Germany at both investigated time points. The seroprevalence at the state level fluctuated to some degree, but the overall percentage remained stable, as is to be expected for an endemic pestivirus lacking any form of control measures.

In Vivo Characterization of Tick-Borne Encephalitis Virus in Bank Voles (Myodes glareolus).

Tick-borne encephalitis is the most important tick-transmitted zoonotic virus infection in Eurasia, causing severe neurological symptoms in humans. The causative agent, the tick-borne encephalitis virus (TBEV), circulates between ticks and a variety of mammalian hosts. To study the interaction between TBEV and one of its suspected reservoir hosts, bank voles of the Western evolutionary lineage were inoculated subcutaneously with either one of eight TBEV strains or the related attenuated Langat virus, and were euthanized after 28 days. In addition, a subset of four strains was characterized in bank voles of the Carpathian linage. Six bank voles were inoculated per strain, and were housed together in groups of three with one uninfected in-contact animal each. Generally, most bank voles did not show any clinical signs over the course of infection. However, one infected bank vole died and three had to be euthanized prematurely, all of which had been inoculated with the identical TBEV strain (Battaune 17-H9, isolated in 2017 in Germany from a bank vole). All inoculated animals seroconverted, while none of the in-contact animals did. Viral RNA was detected via real-time RT-PCR in the whole blood samples of 31 out of 74 inoculated and surviving bank voles. The corresponding serum sample remained PCR-negative in nearly all cases (29/31). In addition, brain and/or spine samples tested positive in 11 cases, mostly correlating with a positive whole blood sample. Our findings suggest a good adaption of TBEV to bank voles, combining in most cases a low virulence phenotype with detectable virus replication and hinting at a reservoir host function of bank voles for TBEV.

Exploring the Reservoir Hosts of Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) is an important arbovirus, which is found across large parts of Eurasia and is considered to be a major health risk for humans. Like any other arbovirus, TBEV relies on complex interactions between vectors, reservoir hosts, and the environment for successful virus circulation. Hard ticks are the vectors for TBEV, transmitting the virus to a variety of animals. The importance of these animals in the lifecycle of TBEV is still up for debate. Large woodland animals seem to have a positive influence on virus circulation by providing a food source for adult ticks; birds are suspected to play a role in virus distribution. Bank voles and yellow-necked mice are often referred to as classical virus reservoirs, but this statement lacks strong evidence supporting their highlighted role. Other small mammals (e.g., insectivores) may also play a crucial role in virus transmission, not to mention the absence of any suspected reservoir host for non-European endemic regions. Theories highlighting the importance of the co-feeding transmission route go as far as naming ticks themselves as the true reservoir for TBEV, and mammalian hosts as a mere bridge for transmission. A deeper insight into the virus reservoir could lead to a better understanding of the development of endemic regions. The spatial distribution of TBEV is constricted to certain areas, forming natural foci that can be restricted to sizes of merely 500 square meters. The limiting factors for their occurrence are largely unknown, but a possible influence of reservoir hosts on the distribution pattern of TBE is discussed. This review aims to give an overview of the multiple factors influencing the TBEV transmission cycle, focusing on the role of virus reservoirs, and highlights the questions that are waiting to be further explored.

The Occurrence of a Commercial Npro and Erns Double Mutant BVDV-1 Live-Vaccine Strain in Newborn Calves.

The major source for the spread of bovine viral diarrhea virus (BVDV) are in-utero infected, immunotolerant, persistently infected (PI) animals since they shed enormous amounts of viruses throughout their lives. During the sequence-based virus typing of diagnostic ear notch samples performed in the context of the obligatory German BVDV eradication program, the commercial Npro and Erns double mutant BVDV-1 live-vaccine strain KE-9 was detected in seven newborn calves; their mothers were immunized in the first trimester of gestation. Six calves either succumbed or were culled immediately, but the one remaining animal was closely monitored for six months. The viral RNA was detected in the skin sample taken in its first and fifth week of life, but the virus could not be isolated. Further skin biopsies that were taken at monthly intervals as well as every serum and urine sample, nasal, oral, and rectal swabs taken weekly tested BVDV negative. However, neutralizing titers against BVDV-1 remained at a consistently high level. To further control for virus shedding, a BVDV antibody and antigen negative calf was co-housed which remained negative throughout the study. The missing viremia, a lack of excretion of infectious virus and negative follow-up skin samples combined with consistently high antibody titers speak against the induction of the classical persistent infection by vaccination with recombinant KE-9 during gestation. We, therefore, suggest that the epidemiological impact of the RNA/antigen positivity for an extended period in the skin is very low. The detection of live-vaccine viruses in skin biopsies mainly represents a diagnostic issue in countries that implemented ear notch-based control programs; and KE9-specific RT-PCRs or sequence analysis can be used to identify these animals and avoid culling measures.