Immune Responses and Pathogenesis following Experimental SARS-CoV-2 Infection in Domestic Cats.

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.

Efficient Direct and Limited Environmental Transmission of SARS-CoV-2 Lineage B.1.22 in Domestic Cats.

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 R0 of 2.18 (95% CI = 0.92 to 4.08), a transmission rate parameter ß 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 (R0 > 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.

Engineering potent live attenuated coronavirus vaccines by targeted inactivation of the immune evasive viral deubiquitinase.

Coronaviruses express a papain-like protease (PLpro) that is required for replicase polyprotein maturation and also serves as a deubiquitinating enzyme (DUB). In this study, using a Middle East respiratory syndrome virus (MERS-CoV) PLpro modified virus in which the DUB is selectively inactivated, we show that the PLpro DUB is an important MERS-CoV interferon antagonist and virulence factor. Although the DUB-negative rMERS-CoVMA replicates robustly in the lungs of human dipeptidyl peptidase 4 knock-in (hDPP4 KI) mice, it does not cause clinical symptoms. Interestingly, a single intranasal vaccination with DUB-negative rMERS-CoVMA induces strong and sustained neutralizing antibody responses and sterilizing immunity after a lethal wt virus challenge. The survival of naïve animals also significantly increases when sera from animals vaccinated with the DUB-negative rMERS-CoVMA are passively transferred, prior to receiving a lethal virus dose. These data demonstrate that DUB-negative coronaviruses could be the basis of effective modified live attenuated vaccines.

Intranasal administration of a live-attenuated recombinant newcastle disease virus expressing the SARS-CoV-2 Spike protein induces high neutralizing antibody levels and protects from experimental challenge infection in hamsters.

The emergence of SARS-CoV-2 in December 2019 resulted in the COVID-19 pandemic. Recurring disease outbreaks repeatedly overloaded the public health sector and severely affected the global economy. We developed a candidate COVID-19 vaccine based on a recombinant Newcastle disease virus (NDV) vaccine vector, encoding a pre-fusion stabilized full-length Spike protein obtained from the original SARS-CoV-2 Wuhan isolate. Vaccination of hamsters by intra-muscular injection or intra-nasal instillation induced high neutralizing antibody responses. Intranasal challenge infection with SARS-CoV-2 strain Lelystad demonstrated that both vaccination routes provided partial protection in the upper respiratory tract, and almost complete protection in the lower respiratory tract, as measured by suppressed viral loads and absence of histological lung lesions. Activity wheel measurements demonstrated that animals vaccinated by intranasal inoculation rapidly recovered to normal activity. NDV constructs encoding the spike of SARS-CoV-2 may be attractive candidates for development of intra-nasal COVID-19 booster vaccines.

A Yellow Fever 17D Virus Replicon-Based Vaccine Platform for Emerging Coronaviruses.

The tremendous global impact of the current SARS-CoV-2 pandemic, as well as other current and recent outbreaks of (re)emerging viruses, emphasize the need for fast-track development of effective vaccines. Yellow fever virus 17D (YF17D) is a live-attenuated virus vaccine with an impressive efficacy record in humans, and therefore, it is a very attractive platform for the development of novel chimeric vaccines against various pathogens. In the present study, we generated a YF17D-based replicon vaccine platform by replacing the prM and E surface proteins of YF17D with antigenic subdomains from the spike (S) proteins of three different betacoronaviruses: MERS-CoV, SARS-CoV and MHV. The prM and E proteins were provided in trans for the packaging of these RNA replicons into single-round infectious particles capable of expressing coronavirus antigens in infected cells. YF17D replicon particles expressing the S1 regions of the MERS-CoV and SARS-CoV spike proteins were immunogenic in mice and elicited (neutralizing) antibody responses against both the YF17D vector and the coronavirus inserts. Thus, YF17D replicon-based vaccines, and their potential DNA- or mRNA-based derivatives, may constitute a promising and particularly safe vaccine platform for current and future emerging coronaviruses.

Experimental and field investigations of exposure, replication and transmission of SARS-CoV-2 in pigs in the Netherlands.

In order to assess the risk of SARS-CoV-2 infection, transmission and reservoir development in swine, we combined results of an experimental and two observational studies. First, intranasal and intratracheal challenge of eight pigs did not result in infection, based on clinical signs and PCR on swab and lung tissue samples. Two serum samples returned a low positive result in virus neutralization, in line with findings in other infection experiments in pigs. Next, a retrospective observational study was performed in the Netherlands in the spring of 2020. Serum samples (N =417) obtained at slaughter from 17 farms located in a region with a high human case incidence in the first wave of the pandemic. Samples were tested with protein micro array, plaque reduction neutralization test and receptor-binding-domain ELISA. None of the serum samples was positive in all three assays, although six samples from one farm returned a low positive result in PRNT (titers 40-80). Therefore we conclude that serological evidence for large scale transmission was not observed. Finally, an outbreak of respiratory disease in pigs on one farm, coinciding with recent exposure to SARS-CoV-2 infected animal caretakers, was investigated. Tonsil swabs and paired serum samples were tested. No evidence for infection with SARS-CoV-2 was found. In conclusion, Although in both the experimental and the observational study few samples returned low antibody titer results in PRNT infection with SARS-CoV-2 was not confirmed. It was concluded that sporadic infections in the field cannot be excluded, but large-scale SARS-CoV-2 transmission among pigs is unlikely.

Predictive Value of Precision-Cut Lung Slices for the Susceptibility of Three Animal Species for SARS-CoV-2 and Validation in a Refined Hamster Model.

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.

Animal models for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.

SARS-CoV-2 infection in farmed minks, the Netherlands, April and May 2020.

Respiratory disease and increased mortality occurred in minks on two farms in the Netherlands, with interstitial pneumonia and SARS-CoV-2 RNA in organ and swab samples. On both farms, at least one worker had coronavirus disease-associated symptoms before the outbreak. Variations in mink-derived viral genomes showed between-mink transmission and no infection link between the farms. Inhalable dust contained viral RNA, indicating possible exposure of workers. One worker is assumed to have attracted the virus from mink.