Large-scale study on virological and serological prevalence of SARS-CoV-2 in cats and dogs in Spain.

The disease produced by the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is currently one of the primary concerns worldwide. Knowing the zoonotic origin of the disease and that several animal species, including dogs and cats, are susceptible to viral infection, it is critical to assess the relevance of pets in this pandemic. Here, we performed a large-scale study on SARS-CoV-2 serological and viral prevalence in cats and dogs in Spain in order to elucidate their role and susceptibility. Samples from animals in contact with COVID-19 positive people and/or compatible symptoms (n = 492), as well as from random animals (n = 1024), were taken. Despite the large number of animals analyzed, only 12 animals (eight dogs and four cats), which represents 0.79% of the total analyzed animals (n = 1516), were positive for viral SARS-CoV-2 RNA detection by reverse transcription quantitative PCR (RT-qPCR) in which viral isolation was possible in four animals. We detected neutralizing antibodies in 34 animals, four of them were also positive for PCR. This study evidences that pets are susceptible to SARS-CoV-2 infection in natural conditions but at a low level, as evidenced by the low percentage of positive animals detected, being infected humans the main source of infection. However, the inclusion of animals in the surveillance of COVID-19 is still recommended.

CELL-LEVEL EXPRESSION OF SARS-COV-2 CELL ENTRY FACTORS IN HUMAN ENDOMETRIUM DURING THE PRECONCEPTION PERIOD

Objective: ACE2 enzyme serves as SARS-CoV-2 human receptor through binding of the viral S protein and subsequent trimming of S protein between S1 and S2 units by host serine proteases as TMPRSS2, CTSB or CTSL. Here, we aim to investigate the expression of the different cell entry proteins involved in SARS-CoV-2 infection in the different cell types of the human endometrium throughout the menstrual cycle using single-cell RNAseq (scRNAseq). Design: Gene expression patterns for SARS-CoV-2 entry molecules were analyzed by scRNAseq in a total of 73,181 endometrial single cells obtained from endometrial biopsies from 19 reproductive-age women across the full menstrual cycle (Fluidigm C1: 2,149 cells) and 10 women from the same cohort (10x: 71,032 cells). For two women, both C1 and 10x data were collected as anchors for comparison. Materials and Methods: After tissue dissociation, single cell capture was performed on Fluidigm C1 system (n= 2,149 cells) or Chromium 10x system (Chromium Next GEM Chip G, 10x Genomics) (n= 71,032 cells) followed by reverse-transcription, cDNA generation and library construction. Barcoded libraries were sequenced in pair-end reads on Nextseq (Illumina) for the C1 dataset or Novaseq (Illumina) for the 10x dataset. Data pre-processing, quality filtering, and statistical analyses were performed using custom Python, R, and Java scripts. Results: Expression analysis across the menstrual cycle showed no significant expression of ACE2 in stromal or unciliated epithelial cells in any cycle phase. TMPRSS2 was expressed more highly in glandular epithelial cells during the early proliferative phase and towards the end of the cycle. Interestingly, expression of CTSB and CTSL was observed in both stromal and epithelial cells across all phases of the menstrual cycle, with CTSB the more abundant of the two. All four genes were simultaneously expressed in less than 0.7% of glandular epithelial cells. Expression analysis during the secretory phase did not detect significant expression of ACE2 (less than 2% of epithelial or stromal cells). TMPRSS2 showed mild expression in about 12% of unciliated epithelial cells. In contrast, CTSB and CTSLwere highly expressed in ∼80% and ∼40% of cells during the mid-late secretory phase, aligning with what we detected via Fluidigm. In addition, while CTSB was highly expressed in both epithelial and stromal cells, CTSL was more highly expressed in stromal cells across the menstrual cycle. Conclusions: Percentages of endometrial cells expressing ACE2, TMPRSS2, CTSB, or CTSL were <2%, ∼12%, ∼80%, and ∼40%, respectively, with <0.7% of cells expressing all four. This finding implies low efficiency of SARS-CoV-2 infection in the endometrium before embryo implantation, providing information to assess preconception endometrial infection risk in COVID-19 asymptomatic carriers. FV & WW contributed equally.

Detection of environmental SARS-CoV-2 RNA in a high prevalence setting in Spain.

Since March 2020, Spain (along with many other countries) has been severely affected by the ongoing coronavirus disease 19 (COVID-19) pandemic caused by the rapid spread of a new virus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2). As part of global efforts to improve disease surveillance, we investigated how readily SARS-CoV-2 RNA could be detected in environmental samples collected from an isolated rural community in Spain with a high COVID-19 prevalence (6% of the population of 883 inhabitants). The first diagnosis of COVID-19-compatible symptoms in the village was recorded on 3 March 2020, and the last known active case resolved on 5 June 2020. By 15 May, two months after strict movement constraints were imposed ('lockdown'), and the cumulative number of symptomatic cases had increased to 53. Of those cases, 22 (41%) had been tested and confirmed by RT-PCR. On 13 May and 5 June, samples were collected from high-use surfaces and clothes in the homes of 13 confirmed cases, from surfaces in nine public service sites (e.g. supermarket and petrol station) and from the wastewater of the village sewage system. SARS-CoV-2 RNA was detected in 7 of 57 (12%) samples, including three households and three public sites. While there is not yet sufficient evidence to recommend environmental surveillance as a standard approach for COVID-19 epidemiology, environmental surveillance research may contribute to advance knowledge about COVID-19 by further elucidating virus shedding dynamics and environmental contamination, including the potential identification of animal reservoirs.