Surveillance for SARS-CoV-2 in Norway Rats (Rattus norvegicus) from Southern Ontario

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from wildlife has raised concerns about spillover from humans to animals, the establishment of novel wildlife reservoirs, and the potential for future outbreaks caused by variants of wildlife origin. Norway rats (Rattus norvegicus) are abundant in urban areas and live in close proximity to humans, providing the opportunity for spillover of SARS-CoV-2. Evidence of SARS-CoV-2 infection and exposure has been reported in Norway rats. We investigated SARS-CoV-2 infection and exposure in Norway rats from Southern Ontario, Canada. From October 2019 to June 2021, 224 rats were submitted by collaborating pest control companies. The majority of samples were collected in Windsor (79.9%;n = 179), Hamilton (13.8%;n = 31), and the Greater Toronto Area (5.8%;n = 13). Overall, 50.0% (n = 112) were female and most rats were sexually mature (55.8%;n = 125). Notably, 202 samples were collected prior to the emergence of variants of concern (VOC) and 22 were collected while the Alpha variant (B.1.1.7) was the predominant circulating VOC in humans. Nasal turbinate (n = 164) and small intestinal (n = 213) tissue samples were analyzed for SARS-CoV-2 RNA by RT-PCR. Thoracic cavity fluid samples (n = 213) were tested for neutralizing antibodies using a surrogate virus neutralization test (sVNT) (GenScript cPass);confirmatory plaque reduction neutralization test (PRNT) was conducted on presumptive positive samples. We did not detect SARS-CoV-2 RNA in any samples tested. Two out of eleven samples positive on sVNT had neutralizing antibodies confirmed positive by PRNT (1 : 40 and 1 : 320 PRNT70);both were collected prior to the emergence of VOC. It is imperative that efforts to control and monitor SARS-CoV-2 include surveillance of rats and other relevant wildlife species as novel variants continue to emerge.

Zooanthroponotic transmission of SARS-CoV-2 and host-specific viral mutations revealed by genome-wide phylogenetic analysis.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a generalist virus, infecting and evolving in numerous mammals, including captive and companion animals, free-ranging wildlife, and humans. Transmission among non-human species poses a risk for the establishment of SARS-CoV-2 reservoirs, makes eradication difficult, and provides the virus with opportunities for new evolutionary trajectories, including the selection of adaptive mutations and the emergence of new variant lineages. Here, we use publicly available viral genome sequences and phylogenetic analysis to systematically investigate the transmission of SARS-CoV-2 between human and non-human species and to identify mutations associated with each species. We found the highest frequency of animal-to-human transmission from mink, compared with lower transmission from other sampled species (cat, dog, and deer). Although inferred transmission events could be limited by sampling biases, our results provide a useful baseline for further studies. Using genome-wide association studies, no single nucleotide variants (SNVs) were significantly associated with cats and dogs, potentially due to small sample sizes. However, we identified three SNVs statistically associated with mink and 26 with deer. Of these SNVs, ~⅔ were plausibly introduced into these animal species from local human populations, while the remaining ~⅓ were more likely derived in animal populations and are thus top candidates for experimental studies of species-specific adaptation. Together, our results highlight the importance of studying animal-associated SARS-CoV-2 mutations to assess their potential impact on human and animal health.

Divergent SARS-CoV-2 variant emerges in white-tailed deer with deer-to-human transmission.

Wildlife reservoirs of broad-host-range viruses have the potential to enable evolution of viral variants that can emerge to infect humans. In North America, there is phylogenomic evidence of continual transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (Odocoileus virginianus) through unknown means, but no evidence of transmission from deer to humans. We carried out an observational surveillance study in Ontario, Canada during November and December 2021 (n = 300 deer) and identified a highly divergent lineage of SARS-CoV-2 in white-tailed deer (B.1.641). This lineage is one of the most divergent SARS-CoV-2 lineages identified so far, with 76 mutations (including 37 previously associated with non-human mammalian hosts). From a set of five complete and two partial deer-derived viral genomes we applied phylogenomic, recombination, selection and mutation spectrum analyses, which provided evidence for evolution and transmission in deer and a shared ancestry with mink-derived virus. Our analysis also revealed an epidemiologically linked human infection. Taken together, our findings provide evidence for sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

SARS-CoV-2 Seroconversion in an Adult Horse with Direct Contact to a COVID-19 Individual.

The authors report on a possible direct exposure to SARS-CoV-2 from a COVID-19-positive individual to an adult horse. The individual, diagnosed with COVID-19 (Delta B.1.617.2), had daily contact to her two horses prior to and during the development of clinical disease. None of the two horses developed abnormal clinical signs or had detectable SARS-CoV-2 in blood, nasal secretion, or feces via RT-qPCR. However, one of the two horses showed close temporal seroconversion to SARS-CoV-2 using a protein-based ELISA and the plaque reduction neutralization test. The results suggest that horses can become silently infected with SARS-CoV-2 following close contact with humans infected with SARS-CoV-2. As a precautionary measure, humans infected with SARS-CoV-2 should avoid close contact with equids and other companion animals during the time of their illness to prevent viral transmission.

Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus.

CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CRISPR RNA (crRNA) containing a "spacer" sequence that specifically complements with the target nucleic acid sequence guides the activation of a CRISPR effector protein (Cas13a, Cas12a or Cas12b), leading to collateral cleavage of RNA or DNA reporters and enormous signal amplification. CRISPR function can be disrupted by some types of sequence mismatches between the spacer and target, according to previous studies. This poses a potential challenge in the detection of variable targets such as RNA viruses with a high degree of sequence diversity, since mismatches can result from target variations. To cover viral diversity, we propose in this study that during crRNA synthesis mixed nucleotide types (degenerate sequences) can be introduced into the spacer sequence positions corresponding to viral sequence variations. We test this crRNA design strategy in the context of the Cas13a-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean-Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic distribution and broad sequence variability. The degenerate-sequence CRISPR diagnostic proves functional, sensitive, specific and rapid. It detects within 30-40 minutes 1 copy/µl of viral RNA from CCHFV strains representing all clades, and from more recently identified strains with new mutations in the CRISPR target region. Also importantly, it shows no cross-reactivity with a variety of CCHFV-related viruses. This proof-of-concept study demonstrates that the degenerate sequence-based CRISPR diagnostic is a promising tool of choice for effective detection of highly variable viral pathogens.

Susceptibility of turkeys, chickens and chicken embryos to SARS-CoV-2.

The susceptibility of turkeys, chickens and chicken embryos to SARS-CoV-2 was evaluated by experimental infection. Turkeys and chickens were inoculated using a combination of intranasal, oral and ocular routes. Both turkeys and chickens did not develop clinical disease or seroconvert following inoculation. Viral RNA was not detected in oral swabs, cloacal swabs or in tissues using quantitative real-time RT-PCR. In addition, chicken embryos were inoculated by various routes including the yolk sac, intravenous, chorioallantoic membrane and allantoic cavity. In all instances, chicken embryos failed to support replication of the virus. SARS-CoV-2 does not affect turkeys or chickens in the current genetic state and does not pose any potential risk to establish an infection in both species of domestic poultry.

A glucose meter interface for point-of-care gene circuit-based diagnostics.

Recent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5' untranslated region (5' UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.