Wastewater-based surveillance (WBS) has shown to be an effective tool in monitoring the spread of SARS-CoV-2 and has helped guide public health actions. Consequently, WBS has expanded to now include the monitoring of mpox virus (MPXV) to contribute to its mitigation efforts. In this study, we demonstrate a unique sample processing and a molecular diagnostic strategy for MPXV detection that can inform on the epidemiological situation of mpox outbreaks through WBS. We conducted WBS for MPXV in 22 Canadian wastewater treatment plants (WWTPs) for 14 weeks. Three MPXV qPCR assays were assessed in this study for the detection of MPXV which include the G2R assays (G2R_WA and G2R_G) developed by the Centers for Disease Control and Prevention (CDC) in 2010, and an in-house-developed assay that we have termed G2R_NML. The G2R_NML assay was designed using reference genomes from the 2022 MPXV outbreak and provides a larger qPCR amplicon size to facilitate Sanger sequencing. Results show that all three assays have similar limits of detection and are able to detect the presence of MPXV in wastewater. The G2R_NML assay produced a significantly greater number of Sanger sequence-confirmed MPXV results compared to the CDC G2R assays. Detection of MPXV was possible where provincial surveillance indicated overall low caseloads, and in some sites forewarning of up to several weeks was observed. Overall, this study proposes that WBS of MPXV provides additional information to help fill knowledge gaps in clinical case-surveillance and is potentially an essential component to the management of mpox.

Résumé Contexte : Les variants préoccupants du coronavirus 2 du syndrome respiratoire aigu sévère (SRAS-CoV-2) sont associés à une augmentation de l’infectivité, de la gravité et de la mortalité de la maladie à coronavirus 2019 (COVID-19) et sont de plus en plus souvent détectés dans le cadre de la surveillance clinique et de la surveillance des eaux usées au Canada et dans le reste du monde. Dans cette étude, nous présentons un essai de réaction en chaîne quantitative de la polymérase après la transcription inverse en temps réel (RT-qPCR) pour la détection de la mutation D377Y du gène N associée au variant Delta du SRAS-CoV-2 dans les eaux usées. Méthodes : Des échantillons d’eaux usées (n = 980) ont été prélevés dans six villes et 17 collectivités rurales du Canada de juillet à novembre 2021 et ont fait l’objet d’un dépistage de la mutation D377Y. Résultats : Le variant Delta a été détecté dans toutes les grandes villes canadiennes et les régions éloignées du Nord, ainsi que dans la moitié des collectivités rurales du sud. La sensibilité et la spécificité de cet essai étaient suffisantes pour détecter et quantifier le variant Delta dans les eaux usées afin de contribuer à un dépistage et à une surveillance rapides au niveau de la population. Conclusion : Cette étude démontre un nouveau test RT-qPCR rentable pour suivre la

Background:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are associated with increased infectivity, severity, and mortality of coronavirus disease 2019 (COVID-19) and have been increasingly detected in clinical and wastewater surveillance in Canada and internationally. In this study, we present a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay for detection of the N gene D377Y mutation associated with the SARS-CoV-2 Delta variant in wastewater.Methods:Wastewater samples (n=980) were collected from six cities and 17 rural communities across Canada from July to November 2021 and screened for the D377Y mutation.Results:The Delta variant was detected in all major Canadian cities and northern remote regions, and half of the southern rural communities. The sensitivity and specificity of this assay were sufficient for detection and quantitation of the Delta variant in wastewater to aid in rapid population-level screening and surveillance.Conclusion:This study demonstrates a novel cost-effective RT-qPCR assay for tracking the spread of the SARS-CoV-2 Delta variant. This rapid assay can be easily integrated into current wastewater surveillance programs to aid in population-level variant tracking.

The sudden emergence and spread of Monkeypox in non-endemic parts of the world is currently not well understood. Infections are often mis-diagnosed and surveillance strategies are scarce. Wastewater-based surveillance (WBS) of human Monkeypox virus (MPXV) can help supplement our current clinical surveillance and mitigation efforts. WBS has shown to be an effective tool in monitoring the spread of other infectious pathogens, such as SARS-CoV-2 and its variants, and has helped guide public health actions. In this study, we describe how WBS can be used to detect MPXV in wastewater. We conducted WBS for MPXV in 22 wastewater treatment plants (WWTPs) over a period of 14 weeks. Nucleic acids were extracted using the MagAttract PowerMicrobiome DNA/RNA extraction kit. Three real-time qPCR assays were assessed for the detection of MPXV in wastewater. These included the G2R assays (G2R\_WA and G2R\_G) developed by the Centers for Disease Control and Prevention (CDC) in 2010, as well as an in-house-developed assay (G2R\_NML). The G2R\_G (generic) assay was designed to detect both the Congo and West African clades (re-named to Clades one and two, respectively) of viruses while the G2R\_WA assay was designed to detect the West African clade (Clade one). The G2R\_NML assay was designed using reference genomes of the 2022 MPXV outbreak. Our results show that all three assays have similar limits of detection and are all able to detect the presence of MPXV in wastewater. Following detection through real time qPCR, Sanger sequencing was performed on the resulting amplicon products, with the assembled contigs then undergoing analysis using nucleotide Basic Local Alignment Search Tool (BLAST). Due in part to the longer amplicon size of the G2R_NML assay, a significantly greater number of positive detections were identified as originating from MPXV compared to the CDC G2R assays. The ability to detect trace amounts of MPXV in wastewater as well as obtain Sanger sequence confirmation, has allowed for the successful surveillance of this virus in wastewater.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis study was funded by internal funds (Government of Canada)### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

SARS-CoV-2 variants of concern (VoC) have been increasingly detected in clinical surveillance in Canada and internationally. These VoC are associated with higher transmissibility rates and in some cases, increased mortality. In this work we present a national wastewater survey of the distribution of three SARS-CoV-2 mutations found in the B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma) VoC, namely the S-gene 69-70 deletion, N501Y mutation, and N-gene D3L. RT-qPCR allelic discrimination assays were sufficiently sensitive and specific for detection and relative quantitation of SARS-CoV-2 variants in wastewater to allow for rapid population-level screening and surveillance. We tested 261 samples collected from 5 Canadian cities (Vancouver, Edmonton, Toronto, Montreal, and Halifax) and 6 communities in the Northwest Territories from February 16th to March 28th, 2021. VoC were not detected in the Territorial communities, suggesting the absence of VoC SARS-CoV-2 cases in those communities. Percentage of variant remained low throughout the study period in the majority of the sites tested, however the Toronto sites showed a marked increase from ~25% to ~75% over the study period. The results of this study highlight the utility of population level molecular surveillance of SARS-CoV-2 VoC using wastewater. Wastewater monitoring for VoC can be a powerful tool in informing public health responses, including monitoring trends independent of clinical surveillance and providing early warning to communities.

The brown pansy, Junonia stygia (Aurivillius, 1894) (Lepidoptera: Nymphalidae), is a widespread West African forest butterfly. Genome skimming by Illumina sequencing allowed assembly of a complete 15,233 bp circular mitogenome from J. stygia consisting of 79.5% AT nucleotides. Mitochondrial gene order and composition is identical to other butterfly mitogenomes. Junonia stygia COX1 features an atypical CGA start codon, while ATP6, COX1, COX2, ND4, and ND4L exhibit incomplete stop codons. Phylogenetic reconstruction supports a monophyletic Subfamily Nymphalinae, Tribe Junoniini, and genus Junonia. The phylogenetic tree places Junonia iphita and J. stygia as basal mitogenome lineages sister to the remaining Junonia sequences.

Foot-and-mouth disease (FMD) is a highly contagious disease that affects cattle, sheep, goats, pigs, and over 70 species of wildlife. FMD continues to be a major economic concern for livestock productivity in many countries. FMDV has seven serotypes O, A, Asia 1, C, and Southern Africa Territories (SAT) 1, 2, and 3. Although SAT 1, and SAT 3 outbreaks are not as common as serotypes O, A, Asia 1, and SAT 2, outbreaks have also been reported. The recent outbreaks of SAT 1 occurred in Cameroon, Zimbabwe, South Africa, and Uganda, while most recent SAT 3 occurred in Namibia in 2019. The development of rapid and easy-to-perform FMDV detection tests is critical to control the outbreak and spread of FMD. The current project has produced monoclonal antibodies (mAb) against FMDV serotypes SAT 1, and SAT 3. Using these mAbs, two lateral flow immunochromatographic (LFI) strip tests for the detection of FMDV SAT 1, and SAT 3 have been developed. SAT 1 strip test detected 14 out of 15 SAT 1 field isolates. The SAT 3 strip test detected all four SAT 3 isolates tested, but the signal is weak for UGA 10/97 and showed no cross-reactivity with other FMDV serotypes. The diagnostic specificities of the SAT 1 and the SAT 3 tests are 100 %, which are higher than double antibody sandwich (DAS) ELISA. The diagnostic sensitivity of the SAT 1 test strip is lower than that of DAS ELISA, while the diagnostic sensitivity of the SAT 3 test strip is similar to that of DAS ELISA. The first reported SAT 1 and SAT 3 strip test combined with the previously developed SAT 2 strip test can be used for quick diagnosis in endemic countries in Africa. Rapid identification of FMDV serotypes is critical for disease control and vaccine selection. Also, these strip tests can be used in the laboratory to quickly screen samples from the field.