Host barriers to SARS-CoV-2 demonstrated by ferrets in a high-exposure domestic setting

Ferrets (Mustela putorius furo) are mustelids of special relevance to laboratory studies of respiratory viruses and have been shown to be susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and onward transmission. Here, we report the results of a natural experiment where 29 ferrets in one home had prolonged, direct contact and constant environmental exposure to two humans with symptomatic disease, one of whom was confirmed positive for SARS-CoV-2. We observed no evidence of SARS-CoV-2 transmission from humans to ferrets based on viral and antibody assays. To better understand this discrepancy in experimental and natural infection in ferrets, we compared SARS-CoV-2 sequences from natural and experimental mustelid infections and identified two surface glycoprotein Spike (S) mutations associated with mustelids. While we found evidence that angiotensin-converting enzyme II provides a weak host barrier, one mutation only seen in ferrets is located in the novel S1/S2 cleavage site and is computationally predicted to decrease furin cleavage efficiency. These data support the idea that host factors interacting with the novel S1/S2 cleavage site may be a barrier in ferret SARS-CoV-2 susceptibility and that domestic ferrets are at low risk of natural infection from currently circulating SARS-CoV-2. We propose two mechanistically grounded hypotheses for mustelid host adaptation of SARS-CoV-2, with possible effects that require additional investigation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is a zoonotic member of Coronaviridae that emerged in 2019 as a major viral pandemic (1). As of February 2021, there have been ∼102 million confirmed COVID-19 cases globally and ∼2.2 million deaths (2). SARS-CoV-2 uses angiotensin I converting enzyme-2 (ACE2) as its primary cellular receptor for host entry and infection (3–5). In silico analyses of ACE2 genes in diverse mammalian species show that residues important to viral binding are moderately conserved between humans and several domestic animals, and a broad range of species have been demonstrated to be permissive to infection in vitro and in vivo (6–10).It is not yet known whether natural infection of animals plays a role in public health epidemiology or has the potential to establish endemic reservoirs and threaten wildlife. SARS-CoV-2 has been observed to be capable of natural human-to-animal reverse zoonoses, transmitting from infected individuals into mink (11), dogs (12), and felines (13–15). American mink (Neovison vison) are currently the only species observed to have natural human-to-animal spillover and onward transmission (11). To date, at least 27 mink farms in The Netherlands, Spain, Denmark, and United States have reported outbreaks, including at least one probable case of mink-to-human transmission (16, 17).SARS-CoV-2 has also been shown to productively infect several species, including ferrets and domestic cats, in vivo (9, 10, 18, 19). Ferrets (Mustela putorius furo) are of special relevance to laboratory studies of respiratory viruses like Influenza A virus and recapitulate clinical pathophysiological aspects of human disease. Given their susceptibility to experimental infection and onward transmission via direct and indirect contact, ferrets have been proposed as an animal model to study SARS-CoV-2 transmission. Based on in vivo data, we expect all naïve ferrets in direct contact with an infected ferret will 1) become infected, 2) have measurable viral shedding or RNA via oral swabs up to 19 d postinfection, and 3) seroconvert with measurable antibodies against SARS-CoV-2 receptor binding domain (RBD) (18, 19).In March 2020, during the first wave of the SARS-CoV-2/COVID-19 pandemic in the New England area, we developed a rapid response study to investigate the potential for human-to-animal spillover and onward transmission in domestic, farm, and wildlife species (CoVERS: Coronavirus Epidemiological Response and Surveillance). The goal of CoVERS is to understand whether and how SARS-CoV-2 transmission is occurring at these interfaces, to refine public health guidelines, investigate whether there are additional risks to animal or human health associated with spillover, and evaluate the potential for establishment of endemic reservoirs. In the CoVERS in-home study, participants are sent a “swab and send” kit, which provides materials and instructions to safely take longitudinal nasal and oral samples from their animals, store them in their freezers, and send them back for viral screening. This community science approach allows wide surveillance with no risk of human transmission, as kits are decontaminated and opened in biosafety cabinets. Here, we highlight one enrolled household that created an exceptional natural experiment with direct relevance to our understanding of SARS-CoV-2 reverse zoonosis and animal models of disease.     

Effectiveness of diclofenac versus paracetamol in knee osteoarthritis: a randomised controlled trial in primary care

Background

The effectiveness of diclofenac versus paracetamol in primary care patients with pain caused by knee osteoarthritis is unclear.

Aim

To assess the effectiveness of diclofenac compared with paracetamol over a period of 2, 4, and 12 weeks in patients with knee osteoarthritis.

Design and setting

Randomised controlled trial in general practice.

Method

There were 104 patients included in the study, they were aged ≥45 years consulting their GP with knee pain caused by knee osteoarthritis. Patients were randomly allocated to diclofenac (n = 52) or paracetamol (n = 52) for at least 2 weeks. Primary outcomes were daily knee pain severity, and knee pain and function measured with the Knee Injury and Osteoarthritis Outcome Score (KOOS).

Results

Over a period of 2- and 4-weeks follow-up, no significant difference in daily knee pain was found between the patient groups: estimated differences of 0.5 (95% CI = −0.2 to 1.3) and −0.2 (95% CI = −1.0 to 0.7), respectively. Over the 12-weeks follow-up, no significant differences were found between both groups for KOOS pain: estimated difference of −2.8 (95% CI = −10.7 to 5.1) and KOOS function of −2.7 (−10.6 to 5.0).

Conclusion

Over a period of 2- and 4-weeks follow-up no significant difference in daily measured knee pain severity was found between primary care patients with knee osteoarthritis taking paracetamol or diclofenac. Also, over a period of 12-weeks follow-up no significant differences were found regarding KOOS pain and KOOS function between both groups. Patients more frequently reported minor adverse events after taking diclofenac (64%) than paracetamol (46%).     

Phospholipase A2 levels in acute chest syndrome of sickle cell disease

Acute chest syndrome (ACS) is associated with significant morbidity and is the leading cause of death in patients with sickle cell disease (SCD). Recent reports suggest that bone marrow fat embolism can be detected in many cases of severe ACS. Secretory phospholipase A2 (sPLA2) is an important inflammatory mediator and liberates free fatty acids, which are felt to be responsible for the acute lung injury of the fat embolism syndrome. We measured SPLA2 levels in 35 SCD patients during 20 admissions for ACS, 10 admissions for vaso-occlusive crisis, and during 12 clinic visits when patients were at the steady state. Eleven non-SCD patients with pneumonia were also evaluated. To determine if there was a relationship between sPLA2 and the severity of ACS we correlated SPLA2 levels with the clinical course of the patient. In comparison with normal controls (mean = 3.1 +/- 1.1 ng/mL), the non- SCD patients with pneumonia (mean = 68.6 +/- 82.9 ng/mL) and all three SCD patient groups had an elevation of SPLA2 (steady state mean = 10.0 +/- 8.4 ng/mL; vaso-occlusive crisis mean = 23.7 +/- 40.5 ng/mL; ACS mean = 336 +/- 209 ng/mL). In patients with ACS sPLA2 levels were 100- fold greater than normal control values, 35 times greater than values in SCD patients at baseline, and five times greater than non-SCD patients with pneumonia. The degree of SPLA2 elevation in ACS correlated with three different measures of clinical severity and, in patients followed sequentially, the rise in SPLA2 coincided with the onset of ACS. The dramatic elevation of SPLA2 in patients with ACS but not in patients with vaso-occlusive crisis or non-SCD patients with pneumonia and the correlation between levels of SPLA2 and clinical severity suggest a role for SPLA2 in the diagnosis and, perhaps, in the pathophysiology of patients with ACS.     

Effects of normal platelets on proliferation and constitutive cytokine secretion by human acute myelogenous leukaemia blasts

The effects of soluble E-selectin, P-selectin and normal platelets on acute myelogenous leukaemia (AM L) blasts were investigated in vitro. We investigated effects on spontaneous and cytokine-dependent blast proliferation, and constitutive blast secretion of different cytokines. The presence of normal platelets during in vitro culture caused a dose-dependent increase in both spontaneous and cytokine-dependent AML blast proliferation. Addition of platelets also increased constitutive blast secretion of Interleukin 1beta (IL1beta ), IL6, GM-CSF and TNFalpha, whereas platelets had no effect on the release of IL1 receptor antagonist. The effects of platelets on constitutive cytokine secretion were also detected when platelets and AML blasts were cultured in different chambers separated by a permeable membrane, and a further enhancement was achieved when blasts and platelets were cultured together. Soluble P-selectin had no effect on constitutive AML blast cytokine secretion or the platelet-induced enhancement of the secretion. However, both soluble E- and P-selectin altered AML blast proliferation for a minority of patients. We conclude that normal platelets can modulate the function of human AML blasts in vitro.     

Age-dependent neurological phenotypes in a mouse model of PRRT2-related diseases

Paroxysmal kinesigenic dyskinesia is an episodic movement disorder caused by dominant mutations in the proline-rich transmembrane protein PRRT2, with onset in childhood and typically with improvement or resolution by middle age. Mutations in the same gene may also cause benign infantile seizures, which begin in the first year of life and typically remit by the age of 2 years. Many details of PRRT2 function at the synapse, and the effects of mutations on neuronal excitability in the pathophysiology of epilepsy and dyskinesia, have emerged through the work of several groups over the last decade. However, the age dependence of the phenotypes has not been explored in detail in transgenic models. Here, we report our findings in heterozygous and homozygous Prrt2 knockout mice that recapitulate the age dependence of dyskinesia seen in the human disease. We show that Prrt2 deletion reduces the levels of synaptic proteins in a dose-dependent manner that is most pronounced at postnatal day 5 (P5), attenuates at P60, and disappears by P180. In a test for foot slippage while crossing a balance beam, transient loss of coordination was most pronounced at P60 and less prominent at age extremes. Slower traverse time was noted in homozygous knockout mice only, consistent with the ataxia seen in rare individuals with biallelic loss of function mutations in Prrt2. We thus identify three age-dependent phenotypic windows in the mouse model, which recapitulate the pattern seen in humans with PRRT2-related diseases.

     

Graft function 1 year after pregnancy in an islet‐transplanted patient

Pancreatic islet transplantation is a treatment option for patients with type 1 diabetes (T1D), but pregnancy has generally not been advised for women after receiving an islet allograft. We hereby describe what is to our knowledge the first successful pregnancy and persistent graft function in a woman 4 years after her initial islet transplantation. A 37‐year‐old woman with brittle type 1 diabetes was transplanted with two separate islet graft infusions, eventually becoming insulin independent. Ten months after her second transplantation, her immunosuppression was switched from tacrolimus and sirolimus to tacrolimus, azathioprine, and prednisolone, due to her wish to become pregnant. She became pregnant one year later, and after 38 weeks of uncomplicated pregnancy, she gave birth to a healthy child by C‐section. The current report suggests that pregnancy and childbirth can be accomplished after islet transplantation without loss of islet graft function.     

Regulation of erythropoietin production in a human hepatoblastoma cell line

Production of immuno and biologically active erythropoietin was documented to occur in the human hepatoblastoma cell line HepG-2. The expression of the erythropoietin gene was further verified by Northern blot analysis using a single stranded RNA probe. In vitro studies showed that erythropoietin production by these cells was not stimulated by hypoxia or cobalt chloride, but was related to the proliferative activity of the cells in culture. In addition it was found that the secretion of erythropoietin was almost completely abrogated by tunicamycin, an inhibitor of N-linked glycosylation. This effect of tunicamycin was also observed in a permanently transfected cell line that secretes erythropoietin in large quantities.