ß-Cyclodextrins as affordable antivirals to treat coronavirus infection.

The SARS-CoV-2 pandemic made evident that there are only a few drugs against coronavirus. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety profile. Starting from a list of 116 drug candidates, we used molecular modelling tools to rank the 44 most promising inhibitors. Next, we tested their efficacy as antivirals against α and ß coronaviruses, such as the HCoV-229E and SARS-CoV-2 variants. Four drugs, OSW-1, U18666A, hydroxypropyl-ß-cyclodextrin (HßCD) and phytol, showed in vitro antiviral activity against HCoV-229E and SARS-CoV-2. The mechanism of action of these compounds was studied by transmission electron microscopy and by fusion assays measuring SARS-CoV-2 pseudoviral entry into target cells. Entry was inhibited by HßCD and U18666A, yet only HßCD inhibited SARS-CoV-2 replication in the pulmonary Calu-3 cells. Compared to the other cyclodextrins, ß-cyclodextrins were the most potent inhibitors, which interfered with viral fusion via cholesterol depletion. ß-cyclodextrins also prevented infection in a human nasal epithelium model ex vivo and had a prophylactic effect in the nasal epithelium of hamsters in vivo. All accumulated data point to ß-cyclodextrins as promising broad-spectrum antivirals against different SARS-CoV-2 variants and distant alphacoronaviruses. Given the wide use of ß-cyclodextrins for drug encapsulation and their high safety profile in humans, our results support their clinical testing as prophylactic antivirals.

Extended Viral Shedding of MERS-CoV Clade B Virus in Llamas Compared with African Clade C Strain.

Middle East respiratory syndrome coronavirus (MERS-CoV) clade B viruses are found in camelids and humans in the Middle East, but clade C viruses are not. We provide experimental evidence for extended shedding of MERS-CoV clade B viruses in llamas, which might explain why they outcompete clade C strains in the Arabian Peninsula.

Agreement and differential use of laboratory methods for the detection and quantification of SARS-CoV-2 in experimentally infected animals

Rodents are widely used for the development of COVID-19-like animal models, the virological outcome being determined through several laboratory methods reported in the literature. Our objective was to assess the agreement between methods performed on different sample types from 342 rodents experimentally infected with SARS-CoV-2 (289 golden Syrian hamsters and 53 K18-hACE2 mice). Our results showed moderate agreement between methods detecting active viral replication, and that increasing viral loads determined by either RT-qPCR or infectious viral titration corresponded to increasing immunohistochemical scores. The percentage of agreement between methods decreased over experimental time points, and we observed poor agreement between RT-qPCR results and viral titration from oropharyngeal swabs. In conclusion, RT-qPCR and viral titration on tissue homogenates are the most reliable techniques to determine the presence and replication of SARS-CoV-2 in the early and peak phases of infection, and immunohistochemistry is valuable to evaluate viral distribution patterns in the infected tissues.

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.

Protective efficacy of an RBD-based Middle East respiratory syndrome coronavirus (MERS-CoV) particle vaccine in llamas.

Ongoing outbreaks of Middle East respiratory syndrome coronavirus (MERS-CoV) continue posing a global health threat. Vaccination of livestock reservoir species is a recommended strategy to prevent spread of MERS-CoV among animals and potential spillover to humans. Using a direct-contact llama challenge model that mimics naturally occurring viral transmission, we tested the efficacy of a multimeric receptor binding domain (RBD) particle-display based vaccine candidate. While MERS-CoV was transmitted to naïve animals exposed to virus-inoculated llamas, immunization induced robust virus-neutralizing antibody responses and prevented transmission in 1/3 vaccinated, in-contact animals. Our exploratory study supports further improvement of the RBD-based vaccine to prevent zoonotic spillover of MERS-CoV.

First Detection of SARS-CoV-2 Delta (B.1.617.2) Variant of Concern in a Dog with Clinical Signs in Spain.

Several cases of naturally infected dogs with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported despite the apparently low susceptibility of this species. Here, we document the first reported case of infection caused by the Delta (B.1.617.2) variant of concern (VOC) in a dog in Spain that lived with several household members suffering from Coronavirus Infectious Disease 2019 (COVID-19). The animal displayed mild digestive and respiratory clinical signs and had a low viral load in the oropharyngeal swab collected at the first sampling. Whole-genome sequencing indicated infection with the Delta variant, coinciding with the predominant variant during the fifth pandemic wave in Spain. The dog seroconverted, as detected 21 days after the first sampling, and developed neutralizing antibodies that cross-neutralized different SARS-CoV-2 variants. This study further emphasizes the importance of studying the susceptibility of animal species to different VOCs and their potential role as reservoirs in the context of COVID-19.

Chronological brain lesions after SARS-CoV-2 infection in hACE2-transgenic mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes respiratory disease, but it can also affect other organs including the central nervous system. Several animal models have been developed to address different key questions related to Coronavirus Disease 2019 (COVID-19). Wild-type mice are minimally susceptible to certain SARS-CoV-2 lineages (beta and gamma variants), whereas hACE2-transgenic mice succumb to SARS-CoV-2 and develop a fatal neurological disease. In this article, we aimed to chronologically characterize SARS-CoV-2 neuroinvasion and neuropathology. Necropsies were performed at different time points, and the brain and olfactory mucosa were processed for histopathological analysis. SARS-CoV-2 virological assays including immunohistochemistry were performed along with a panel of antibodies to assess neuroinflammation. At 6 to 7 days post inoculation (dpi), brain lesions were characterized by nonsuppurative meningoencephalitis and diffuse astrogliosis and microgliosis. Vasculitis and thrombosis were also present and associated with occasional microhemorrhages and spongiosis. Moreover, there was vacuolar degeneration of virus-infected neurons. At 2 dpi, SARS-CoV-2 immunolabeling was only found in the olfactory mucosa, but at 4 dpi intraneuronal virus immunolabeling had already reached most of the brain areas. Maximal distribution of the virus was observed throughout the brain at 6 to 7 dpi except for the cerebellum, which was mostly spared. Our results suggest an early entry of the virus through the olfactory mucosa and a rapid interneuronal spread of the virus leading to acute encephalitis and neuronal damage in this mouse model.

Pigs are not susceptible to SARS-CoV-2 infection but are a model for viral immunogenicity studies.

Conventional piglets were inoculated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through different routes, including intranasal, intratracheal, intramuscular and intravenous ones. Although piglets were not susceptible to SARS-CoV-2 and lacked lesions or viral RNA in tissues/swabs, seroconversion was observed in pigs inoculated parenterally (intramuscularly or intravenously).

Protection against reinfection with D614- or G614-SARS-CoV-2 isolates in golden Syrian hamster.

Reinfections with SARS-CoV-2 have already been documented in humans, although its real incidence is currently unknown. Besides having a great impact on public health, this phenomenon raises the question of immunity generated by a single infection is sufficient to provide sterilizing/protective immunity to a subsequent SARS-CoV-2 re-exposure. The Golden Syrian hamster is a manageable animal model to explore immunological mechanisms able to counteract COVID-19, as it recapitulates pathological aspects of mild to moderately affected patients. Here, we report that SARS-CoV-2-inoculated hamsters resolve infection in the upper and lower respiratory tracts within seven days upon inoculation with the Cat01 (G614) SARS-CoV-2 isolate. Three weeks after the primary challenge, and despite high titres of neutralizing antibodies, half of the animals were susceptible to reinfection by both identical (Cat01, G614) and variant (WA/1, D614) SARS-CoV-2 isolates. However, upon re-inoculation, only nasal tissues were transiently infected with much lower viral replication than those observed after the first inoculation. These data indicate that a primary SARS-CoV-2 infection is not sufficient to elicit a sterilizing immunity in hamster models but protects against lung disease.

Detection of SARS-CoV-2 in a cat owned by a COVID-19-affected patient in Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, is considered a zoonotic pathogen mainly transmitted human to human. Few reports indicate that pets may be exposed to the virus. The present report describes a cat suffering from severe respiratory distress and thrombocytopenia living with a family with several members affected by COVID-19. Clinical signs of the cat prompted humanitarian euthanasia and a detailed postmortem investigation to assess whether a COVID-19-like disease was causing the condition. Necropsy results showed the animal suffered from feline hypertrophic cardiomyopathy and severe pulmonary edema and thrombosis. SARS-CoV-2 RNA was only detected in nasal swab, nasal turbinates, and mesenteric lymph node, but no evidence of histopathological lesions compatible with a viral infection were detected. The cat seroconverted against SARS-CoV-2, further evidencing a productive infection in this animal. We conclude that the animal had a subclinical SARS-CoV-2 infection concomitant to an unrelated cardiomyopathy that led to euthanasia.