Since the beginning of the Coronavirus Disease-19 (COVID-19) pandemic, multiple Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mutations have been reported and led to the emergence of variants of concern (VOC) with increased transmissibility, virulence or immune escape. In parallel, the observation of viral fecal shedding led to the quantification of SARS-CoV-2 genomes in wastewater, providing information about the dynamics of SARS-CoV-2 infections within a population including symptomatic and asymptomatic individuals. Here, we aimed to adapt a sequencing technique initially designed for clinical samples to apply it to the challenging and mixed wastewater matrix, and hence identify the circulation of VOC at the community level. Composite raw sewage sampled over 24 h in two wastewater-treatment plants (WWTPs) from a city in western France were collected weekly and SARS-CoV-2 quantified by RT-PCR. Samples collected between October 2020 and May 2021 were submitted to whole-genome sequencing (WGS) using the primers and protocol published by the ARTIC Network and a MinION Mk1C sequencer (Oxford Nanopore Technologies, Oxford, United Kingdom). The protocol was adapted to allow near-full genome coverage from sewage samples, starting from ∼5% to reach ∼90% at depth 30. This enabled us to detect multiple single-nucleotide variant (SNV) and assess the circulation of the SARS-CoV-2 VOC Alpha, Beta, Gamma, and Delta. Retrospective analysis of sewage samples shed light on the emergence of the Alpha VOC with detection of first co-occurring signature mutations in mid-November 2020 to reach predominance of this variant in early February 2021. In parallel, a mutation-specific qRT-PCR assay confirmed the spread of the Alpha VOC but detected it later than WGS. Altogether, these data show that SARS-CoV-2 sequencing in sewage can be used for early detection of an emerging VOC in a population and confirm its ability to track shifts in variant predominance.

Infectious Human Norovirus in Seawater

Background. Sindbis virus (SINV) is a mosquito-borne alphavirus found in Eurasia, Africa, and Oceania. Clinical SINV infection is characterized by febrile rash and arthritis and sometimes prolonged arthralgia and myalgia. The pathophysiological mechanisms of musculoskeletal and rheumatic disease caused by SINV are inadequately understood. Methods. We studied the muscle pathology of SINV infection ex vivo by examining a unique muscle biopsy obtained from a patient with chronic myalgia and arthralgia 6 months after acute SINV infection and assessed potential genetic predisposing factors by determining the human leukocyte antigen (HLA) and complement factor C4 genes and proteins. In addition, we performed in vitro SINV infections of primary human myoblasts and myotubes. Results. In the muscle biopsy we found evidence of muscle regeneration due to previous necrotic lesions likely caused by earlier SINV infection. We showed that human myoblasts and myotubes were susceptible in vitro for SINV infection as the cells became immunoreactive for viral antigens and cytopathic effect was observed. The patient was homozygous for HLA-B*35 alleles and heterozygous for HLA-DRB1*01 and HLA-DRB1*03 alleles and had total deficiency of C4B protein. Conclusions. This study provides new insights concerning pathological processes leading to chronic symptoms in SINV infection and demonstrates for the first time the susceptibility of human myogenic cells to SINV infection.

Foodborne viral infections rank among the top 5 causes of disease, with noroviruses and hepatitis A causing the greatest burden globally. Contamination of foods by infected food handlers or through environmental pollution are the main sources of foodborne illness, with a lesser role for consumption of products from infected animals. Viral partial genomic sequencing has been used for more than two decades to track foodborne outbreaks and whole genome or metagenomics next-generation-sequencing (NGS) are new additions to the toolbox of food microbiology laboratories. We discuss developments in the field of targeted and metagenomic NGS, with an emphasis on application in food virology, the challenges and possible solutions towards future routine application.

Bivalve molluscan shellfish have been consumed for centuries. Being filter feeders, they may bioaccumulate some microorganisms present in coastal water, either naturally or through the discharge of human or animal sewage. Despite regulations set up to avoid microbiological contamination in shellfish, human outbreaks still occur. After providing an overview showing their implication in disease, this review aims to highlight the diversity of the bacteria or enteric viruses detected in shellfish species, including emerging pathogens. After a critical discussion of the available methods and their limitations, we address the interest of technological developments using genomics to anticipate the emergence of pathogens. In the coming years, further research needs to be performed and methods need to be developed in order to design the future of surveillance and to help risk assessment studies, with the ultimate objective of protecting consumers and enhancing the microbial safety of bivalve molluscan shellfish as a healthy food.