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.

SARS-CoV-2 in animals: susceptibility of animal species, risk for animal and public health, monitoring, prevention and control.

The epidemiological situation of SARS-CoV-2 in humans and animals is continually evolving. To date, animal species known to transmit SARS-CoV-2 are American mink, raccoon dog, cat, ferret, hamster, house mouse, Egyptian fruit bat, deer mouse and white-tailed deer. Among farmed animals, American mink have the highest likelihood to become infected from humans or animals and further transmit SARS-CoV-2. In the EU, 44 outbreaks were reported in 2021 in mink farms in seven MSs, while only six in 2022 in two MSs, thus representing a decreasing trend. The introduction of SARS-CoV-2 into mink farms is usually via infected humans; this can be controlled by systematically testing people entering farms and adequate biosecurity. The current most appropriate monitoring approach for mink is the outbreak confirmation based on suspicion, testing dead or clinically sick animals in case of increased mortality or positive farm personnel and the genomic surveillance of virus variants. The genomic analysis of SARS-CoV-2 showed mink-specific clusters with a potential to spill back into the human population. Among companion animals, cats, ferrets and hamsters are those at highest risk of SARS-CoV-2 infection, which most likely originates from an infected human, and which has no or very low impact on virus circulation in the human population. Among wild animals (including zoo animals), mostly carnivores, great apes and white-tailed deer have been reported to be naturally infected by SARS-CoV-2. In the EU, no cases of infected wildlife have been reported so far. Proper disposal of human waste is advised to reduce the risks of spill-over of SARS-CoV-2 to wildlife. Furthermore, contact with wildlife, especially if sick or dead, should be minimised. No specific monitoring for wildlife is recommended apart from testing hunter-harvested animals with clinical signs or found-dead. Bats should be monitored as a natural host of many coronaviruses.

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.

Advances and gaps in SARS-CoV-2 infection models.

The global response to Coronavirus Disease 2019 (COVID-19) is now facing new challenges such as vaccine inequity and the emergence of SARS-CoV-2 variants of concern (VOCs). Preclinical models of disease, in particular animal models, are essential to investigate VOC pathogenesis, vaccine correlates of protection and postexposure therapies. Here, we provide an update from the World Health Organization (WHO) COVID-19 modeling expert group (WHO-COM) assembled by WHO, regarding advances in preclinical models. In particular, we discuss how animal model research is playing a key role to evaluate VOC virulence, transmission and immune escape, and how animal models are being refined to recapitulate COVID-19 demographic variables such as comorbidities and age.

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.

The SARS-CoV-2 Reproduction Number R0 in Cats.

Domestic cats are susceptible to SARS-CoV-2 virus infection and given that they are in close contact with people, assessing the potential risk cats represent for the transmission and maintenance of SARS-CoV-2 is important. Assessing this risk implies quantifying transmission from humans-to-cats, from cats-to-cats and from cats-to-humans. Here we quantified the risk of cat-to-cat transmission by reviewing published literature describing transmission either experimentally or under natural conditions in infected households. Data from these studies were collated to quantify the SARS-CoV-2 reproduction number R0 among cats. The estimated R0 was significantly higher than one, hence cats could play a role in the transmission and maintenance of SARS-CoV-2. Questions that remain to be addressed are the risk of transmission from humans-to-cats and cats-to-humans. Further data on household transmission and data on virus levels in both the environment around infected cats and their exhaled air could be a step towards assessing these risks.

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.

Transcriptional Differences between Canine Cutaneous Epitheliotropic Lymphoma and Immune-Mediated Dermatoses.

Canine cutaneous epitheliotropic T-cell lymphoma (CETL) and immune-mediated T-cell predominant dermatoses (IMD) share several clinical and histopathological features, but differ substantially in prognosis. The discrimination of ambiguous cases may be challenging, as diagnostic tests are limited and may prove equivocal. This study aimed to investigate transcriptional differences between CETL and IMD, as a basis for further research on discriminating diagnostic biomarkers. We performed 100bp single-end sequencing on RNA extracted from formalin-fixed and paraffin-embedded skin biopsies from dogs with CETL and IMD, respectively. DESeq2 was used for principal component analysis (PCA) and differential gene expression analysis. Genes with significantly different expression were analyzed for enriched pathways using two different tools. The expression of selected genes and their proteins was validated by RT-qPCR and immunohistochemistry. PCA demonstrated the distinct gene expression profiles of CETL and IMD. In total, 503 genes were upregulated, while 4986 were downregulated in CETL compared to IMD. RT-qPCR confirmed the sequencing results for 5/6 selected genes tested, while the protein expression detected by immunohistochemistry was not entirely consistent. Our study revealed transcriptional differences between canine CETL and IMD, with similarities to human cutaneous lymphoma. Differentially expressed genes are potential discriminatory markers, but require further validation on larger sample collections.

Functional Radiogenetic Profiling Implicates ERCC6L2 in Non-homologous End Joining.

Using genome-wide radiogenetic profiling, we functionally dissect vulnerabilities of cancer cells to ionizing radiation (IR). We identify ERCC6L2 as a major determinant of IR response, together with classical DNA damage response genes and members of the recently identified shieldin and CTC1-STN1-TEN1 (CST) complexes. We show that ERCC6L2 contributes to non-homologous end joining (NHEJ), and it may exert this function through interactions with SFPQ. In addition to causing radiosensitivity, ERCC6L2 loss restores DNA end resection and partially rescues homologous recombination (HR) in BRCA1-deficient cells. As a consequence, ERCC6L2 deficiency confers resistance to poly (ADP-ribose) polymerase (PARP) inhibition in tumors deficient for both BRCA1 and p53. Moreover, we show that ERCC6L2 mutations are found in human tumors and correlate with a better overall survival in patients treated with radiotherapy (RT); this finding suggests that ERCC6L2 is a predictive biomarker of RT response.

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.

Haploid genetic screens identify genetic vulnerabilities to microtubule-targeting agents.

The absence of biomarkers to accurately predict anticancer therapy response remains a major obstacle in clinical oncology. We applied a genome-wide loss-of-function screening approach in human haploid cells to characterize genetic vulnerabilities to classical microtubule-targeting agents. Using docetaxel and vinorelbine, two well-established chemotherapeutic agents, we sought to identify genetic alterations sensitizing human HAP1 cells to these drugs. Despite the fact that both drugs act on microtubules, a set of distinct genes were identified whose disruption affects drug sensitivity. For docetaxel, this included a number of genes with a function in mitosis, while for vinorelbine we identified inactivation of FBXW7, RB1, and NF2, three frequently mutated tumor suppressor genes, as sensitizing factors. We validated these genes using independent knockout clones and confirmed FBXW7 as an important regulator of the mitotic spindle assembly. Upon FBXW7 depletion, vinorelbine treatment led to decreased survival of cells due to defective mitotic progression and subsequent mitotic catastrophe. We show that haploid insertional mutagenesis screens are a useful tool to study genetic vulnerabilities to classical chemotherapeutic drugs by identifying thus far unknown sensitivity factors. These results provide a rationale for investigating patient response to vinca alkaloid-based anticancer treatment in relation to the mutational status of these three tumor suppressor genes, and could in the future lead to the establishment of novel predictive biomarkers or suggest new drug combinations based on molecular mechanisms of drug sensitivity.

New tools for old drugs: Functional genetic screens to optimize current chemotherapy.

Despite substantial advances in the treatment of various cancers, many patients still receive anti-cancer therapies that hardly eradicate tumor cells but inflict considerable side effects. To provide the best treatment regimen for an individual patient, a major goal in molecular oncology is to identify predictive markers for a personalized therapeutic strategy. Regarding novel targeted anti-cancer therapies, there are usually good markers available. Unfortunately, however, targeted therapies alone often result in rather short remissions and little cytotoxic effect on the cancer cells. Therefore, classical chemotherapy with frequent long remissions, cures, and a clear effect on cancer cell eradication remains a corner stone in current anti-cancer therapy. Reliable biomarkers which predict the response of tumors to classical chemotherapy are rare, in contrast to the situation for targeted therapy. For the bulk of cytotoxic therapeutic agents, including DNA-damaging drugs, drugs targeting microtubules or antimetabolites, there are still no reliable biomarkers used in the clinic to predict tumor response. To make progress in this direction, meticulous studies of classical chemotherapeutic drug action and resistance mechanisms are required. For this purpose, novel functional screening technologies have emerged as successful technologies to study chemotherapeutic drug response in a variety of models. They allow a systematic analysis of genetic contributions to a drug-responsive or -sensitive phenotype and facilitate a better understanding of the mode of action of these drugs. These functional genomic approaches are not only useful for the development of novel targeted anti-cancer drugs but may also guide the use of classical chemotherapeutic drugs by deciphering novel mechanisms influencing a tumor's drug response. Moreover, due to the advances of 3D organoid cultures from patient tumors and in vivo screens in mice, these genetic screens can be applied using conditions that are more representative of the clinical setting. Patient-derived 3D organoid lines furthermore allow the characterization of the "essentialome", the specific set of genes required for survival of these cells, of an individual tumor, which could be monitored over the course of treatment and help understanding how drug resistance evolves in clinical tumors. Thus, we expect that these functional screens will enable the discovery of novel cancer-specific vulnerabilities, and through clinical validation, move the field of predictive biomarkers forward. This review focuses on novel advanced techniques to decipher the interplay between genetic alterations and drug response.

Stem Cell-Associated Marker Expression in Canine Hair Follicles.

Functional hair follicle (HF) stem cells (SCs) are crucial to maintain the constant recurring growth of hair. In mice and humans, SC subpopulations with different biomarker expression profiles have been identified in discrete anatomic compartments of the HF. The rare studies investigating canine HF SCs have shown similarities in biomarker expression profiles to that of mouse and human SCs. The aim of our study was to broaden the current repertoire of SC-associated markers and their expression patterns in the dog. We combined analyses on the expression levels of CD34, K15, Sox9, CD200, Nestin, LGR5 and LGR6 in canine skin using RT-qPCR, the corresponding proteins in dog skin lysates, and their expression patterns in canine HFs using immunohistochemistry. Using validated antibodies, we were able to define the location of CD34, Sox9, Keratin15, LGR5 and Nestin in canine HFs and confirm that all tested biomarkers are expressed in canine skin. Our results show similarities between the expression profile of canine, human and mouse HF SC markers. This repertoire of biomarkers will allow us to conduct functional studies and investigate alterations in the canine SC compartment of different diseases, like alopecia or skin cancer with the possibility to extend relevant findings to human patients.

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs.

Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8-dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug-induced apoptosis independently from drug uptake, possibly by impairing VRAC-dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D-containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.