Investigating neutrophil function during SARS-CoV2 infection in human airway epithelial cells from elderly and young individuals

The COVID-19 pandemic caused by the SARS-CoV2 virus poses a global health threat with over 5 million deaths recorded. There is little understanding regarding SARS-CoV2 pathogenesis in the human airways and disease severity increases with age. Neutrophils are white blood cells found in large numbers in the airways of the lungs in severe COVID-19 patients. It is not known whether this influx of neutrophils into the airway has a protective or detrimental effect. We aim to understand the role of neutrophils during COVID-19 pathology, using an experimental infection model of the airway epithelium from the eldelry and children. To do this, we collect nasal airway cells from healthy elderly and children and grow them at air-liquid interface. Once differentiation and ciliation of these cells is reached, we infect the cells with SARS-CoV2 virus and allow neutrophils to migrate from the basolateral (blood) to the apical (air) side of the epithelium, similar to a physiological airway. Using flow cytometric analyses, we measure the expression of activation markers and the number of neutrophils that migrate across the epithelium of different ages in response to SARS-CoV2 infection. Preliminary work shows less viable neutrophils recovered from the elderly epithelium, more activated neutrophils when migrating through the elderly epithelium, as well as increased numbers of neutrophils remaining on the basolateral (blood) side of the elderly epithelium. These findings point to an inflammatory neutrophil phenotype influenced by the damaged elderly epithelium and supports the hypothesis that neutrophils are responsible for the severity of disease.

FCoV Viral Sequences of Systemically Infected Healthy Cats Lack Gene Mutations Previously Linked to the Development of FIP.

Feline infectious peritonitis (FIP)-the deadliest infectious disease of young cats in shelters or catteries-is induced by highly virulent feline coronaviruses (FCoVs) emerging in infected hosts after mutations of less virulent FCoVs. Previous studies have shown that some mutations in the open reading frames (ORF) 3c and 7b and the spike (S) gene have implications for the development of FIP, but mainly indirectly, likely also due to their association with systemic spread. The aim of the present study was to determine whether FCoV detected in organs of experimentally FCoV infected healthy cats carry some of these mutations. Viral RNA isolated from different tissues of seven asymptomatic cats infected with the field strains FCoV Zu1 or FCoV Zu3 was sequenced. Deletions in the 3c gene and mutations in the 7b and S genes that have been shown to have implications for the development of FIP were not detected, suggesting that these are not essential for systemic viral dissemination. However, deletions and single nucleotide polymorphisms leading to truncations were detected in all nonstructural proteins. These were found across all analyzed ORFs, but with significantly higher frequency in ORF 7b than ORF 3a. Additionally, a previously unknown homologous recombination site was detected in FCoV Zu1.

Experimental Infection of Pigs with Recent European Porcine Epidemic Diarrhea Viruses.

Porcine epidemic diarrhea virus (PEDV), belonging to the genus Alphacoronavirus, can cause serious disease in pigs of all ages, especially in suckling pigs. Differences in virulence have been observed between various strains of this virus. In this study, four pigs were inoculated with PEDV from Germany (intestine/intestinal content collected from pigs in 2016) and four pigs with PEDV from Italy (intestine/intestinal material collected from pigs in 2016). The pigs were re-inoculated with the same virus on multiple occasions to create a more robust infection and enhance the antibody responses. The clinical signs and pathological changes observed were generally mild. Two distinct peaks of virus excretion were seen in the group of pigs inoculated with the PEDV from Germany, while only one strong peak was seen for the group of pigs that received the virus from Italy. Seroconversion was seen by days 18 and 10 post-inoculation with PEDV in all surviving pigs from the groups that received the inoculums from Germany and Italy, respectively. Attempts to infect pigs with a swine enteric coronavirus (SeCoV) from Slovakia were unsuccessful, and no signs of infection were observed in the inoculated animals.

Susceptibility of Domestic Goat (Capra aegagrus hircus) to Experimental Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) B.1.351/Beta Variant.

A wide range of animal species are susceptible to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Natural and/or experimental infections have been reported in pet, zoo, farmed and wild animals. Interestingly, some SARS-CoV-2 variants, such as B.1.1.7/Alpha, B.1.351/Beta, and B.1.1.529/Omicron, were demonstrated to infect some animal species not susceptible to classical viral variants. The present study aimed to elucidate if goats (Capra aegagrus hircus) are susceptible to the B.1.351/Beta variant. First, an in silico approach was used to predict the affinity between the receptor-binding domain of the spike protein of SARS-CoV-2 B.1.351/Beta variant and angiotensin-converting enzyme 2 from goats. Moreover, we performed an experimental inoculation with this variant in domestic goat and showed evidence of infection. SARS-CoV-2 was detected in nasal swabs and tissues by RT-qPCR and/or immunohistochemistry, and seroneutralisation was confirmed via ELISA and live virus neutralisation assays. However, the viral amount and tissue distribution suggest a low susceptibility of goats to the B.1.351/Beta variant. Therefore, although monitoring livestock is advisable, it is unlikely that goats play a role as SARS-CoV-2 reservoir species, and they are not useful surrogates to study SARS-CoV-2 infection in farmed animals.

Animal Coronaviruses

Coronaviruses (CoVs) infect diverse animal species and cause respiratory, enteric, hepatic, renal, neurologic, and even systemic diseases. The majority of CoVs have a narrow host specificity, but a few CoVs have a broad range of host specificity. This chapter provides a brief review of animal CoVs, including SARS-CoV-2 of animals for their receptors, host tropism, and pathogenesis in target animals.

Animal Models in Human Adenovirus Research.

Human adenovirus (HAdV) infections cause a wide variety of clinical symptoms, ranging from mild upper respiratory tract disease to lethal outcomes, particularly in immunocompromised individuals. To date, neither widely available vaccines nor approved antiadenoviral compounds are available to efficiently deal with HAdV infections. Thus, there is a need to thoroughly understand HAdV-induced disease, and for the development and preclinical evaluation of HAdV therapeutics and/or vaccines, and consequently for suitable standardizable in vitro systems and animal models. Current animal models to study HAdV pathogenesis, persistence, and tumorigenesis include rodents such as Syrian hamsters, mice, and cotton rats, as well as rabbits. In addition, a few recent studies on other species, such as pigs and tree shrews, reported promising data. These models mimic (aspects of) HAdV-induced pathological changes in humans and, although they are relevant, an ideal HAdV animal model has yet to be developed. This review summarizes the available animal models of HAdV infection with comprehensive descriptions of virus-induced pathogenesis in different animal species. We also elaborate on rodent HAdV animal models and how they contributed to insights into adenovirus-induced cell transformation and cancer.

Postmortem Stability of SARS-CoV-2 in Mouse Lung Tissue.

The infectivity of severe acute respiratory syndrome coronavirus 2 in deceased persons and organisms remains unclear. We studied transgenic K18 hACE2 mice to determine the kinetics of virus infectivity after host death. Five days after death, virus infectivity in the lung declined by >96% and RNA copies declined by 48.2%.

Peridomestic Mammal Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Wild animals have been implicated as the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but it is largely unknown how the virus affects most wildlife species and if wildlife could ultimately serve as a reservoir for maintaining the virus outside the human population. We show that several common peridomestic species, including deer mice, bushy-tailed woodrats, and striped skunks, are susceptible to infection and can shed the virus in respiratory secretions. In contrast, we demonstrate that cottontail rabbits, fox squirrels, Wyoming ground squirrels, black-tailed prairie dogs, house mice, and racoons are not susceptible to SARS-CoV-2 infection. Our results expand the knowledge base of susceptible species and provide evidence that human-wildlife interactions could result in continued transmission of SARS-CoV-2.

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.

Anthropogenic Infection of Cats during the 2020 COVID-19 Pandemic.

COVID-19 is a severe acute respiratory syndrome (SARS) caused by a new coronavirus (CoV), SARS-CoV-2, which is closely related to SARS-CoV that jumped the animal-human species barrier and caused a disease outbreak in 2003. SARS-CoV-2 is a betacoronavirus that was first described in 2019, unrelated to the commonly occurring feline coronavirus (FCoV) that is an alphacoronavirus associated with feline infectious peritonitis (FIP). SARS-CoV-2 is highly contagious and has spread globally within a few months, resulting in the current pandemic. Felids have been shown to be susceptible to SARS-CoV-2 infection. Particularly in the Western world, many people live in very close contact with their pet cats, and natural infections of cats in COVID-19-positive households have been described in several countries. In this review, the European Advisory Board on Cat Diseases (ABCD), a scientifically independent board of experts in feline medicine from 11 European Countries, discusses the current status of SARS-CoV infections in cats. The review examines the host range of SARS-CoV-2 and human-to-animal transmissions, including infections in domestic and non-domestic felids, as well as mink-to-human/-cat transmission. It summarises current data on SARS-CoV-2 prevalence in domestic cats and the results of experimental infections of cats and provides expert opinions on the clinical relevance and prevention of SARS-CoV-2 infection in cats.

Experimental Infection of Cattle with SARS-CoV-2.

We inoculated 6 cattle with severe acute respiratory syndrome coronavirus 2 and kept them together with 3 uninoculated cattle. We observed viral replication and specific seroreactivity in 2 inoculated animals, despite high levels of preexisting antibody titers against a bovine betacoronavirus. The in-contact animals did not become infected.

Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached nearly every country in the world with extraordinary person-to-person transmission. The most likely original source of the virus was spillover from an animal reservoir and subsequent adaptation to humans sometime during the winter of 2019 in Wuhan Province, China. Because of its genetic similarity to SARS-CoV-1, it is probable that this novel virus has a similar host range and receptor specificity. Due to concern for human-pet transmission, we investigated the susceptibility of domestic cats and dogs to infection and potential for infected cats to transmit to naive cats. We report that cats are highly susceptible to infection, with a prolonged period of oral and nasal viral shedding that is not accompanied by clinical signs, and are capable of direct contact transmission to other cats. These studies confirm that cats are susceptible to productive SARS-CoV-2 infection, but are unlikely to develop clinical disease. Further, we document that cats developed a robust neutralizing antibody response that prevented reinfection following a second viral challenge. Conversely, we found that dogs do not shed virus following infection but do seroconvert and mount an antiviral neutralizing antibody response. There is currently no evidence that cats or dogs play a significant role in human infection; however, reverse zoonosis is possible if infected owners expose their domestic pets to the virus during acute infection. Resistance to reinfection holds promise that a vaccine strategy may protect cats and, by extension, humans.