A multiplex-NGS approach to identifying respiratory RNA viruses during the COVID-19 pandemic.

A methodological approach based on reverse transcription (RT)-multiplex PCR followed by next-generation sequencing (NGS) was implemented to identify multiple respiratory RNA viruses simultaneously. A convenience sampling from respiratory surveillance and SARS-CoV-2 diagnosis in 2020 and 2021 in Montevideo, Uruguay, was analyzed. The results revealed the cocirculation of SARS-CoV-2 with human rhinovirus (hRV) A, B and C, human respiratory syncytial virus (hRSV) B, influenza A virus, and metapneumovirus B1. SARS-CoV-2 coinfections with hRV or hRSV B and influenza A virus coinfections with hRV C were identified in adults and/or children. This methodology combines the benefits of multiplex genomic amplification with the sensitivity and information provided by NGS. An advantage is that additional viral targets can be incorporated, making it a helpful tool to investigate the cocirculation and coinfections of respiratory viruses in pandemic and post-pandemic contexts.

Detection and genome characterisation of SARS-CoV-2 P.6 lineage in dogs and cats living with Uruguayan COVID-19 patients.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in domestic animals have occurred from the beginning of the pandemic to the present time. Therefore, from the perspective of One Health, investigating this topic is of global scientific and public interest. OBJECTIVES: The present study aimed to determine the presence of SARS-CoV-2 in domestic animals whose owners had coronavirus disease 2019 (COVID-19). METHODS: Nasopharyngeal and faecal samples were collected in Uruguay. Using quantitative polymerase chain reaction (qPCR), we analysed the presence of the SARS-CoV-2 genome. Complete genomes were obtained using ARTIC enrichment and Illumina sequencing. Sera samples were used for virus neutralisation assays. FINDINGS: SARS-CoV-2 was detected in an asymptomatic dog and a cat. Viral genomes were identical and belonged to the P.6 Uruguayan SARS-CoV-2 lineage. Only antiserum from the infected cat contained neutralising antibodies against the ancestral SARS-CoV-2 strain and showed cross-reactivity against the Delta but not against the B.A.1 Omicron variant. MAIN CONCLUSIONS: Domestic animals and the human SARS-CoV-2 P.6 variant comparison evidence a close relationship and gene flow between them. Different SARS-CoV-2 lineages infect dogs and cats, and no specific variants are adapted to domestic animals. This first record of SARS-CoV-2 in domestic animals from Uruguay supports regular surveillance of animals close to human hosts.

Carbohydrate vitrification in aerosolized saliva is associated with the humidity-dependent infectious potential of airborne coronavirus.

An accepted murine analogue for the environmental behavior of human SARS coronaviruses was aerosolized in microdroplets of its culture media and saliva to observe the decay of its airborne infectious potential under relative humidity (RH) conditions relevant to conditioned indoor air. Contained in a dark, 10 m3 chamber maintained at 22°C, murine hepatitis virus (MHV) was entrained in artificial saliva particles that were aerosolized in size distributions that mimic SARS-CoV-2 virus expelled from infected humans' respiration. As judged by quantitative PCR, more than 95% of the airborne MHV aerosolized was recovered from microdroplets with mean aerodynamic diameters between 0.56 and 5.6 µm. As judged by its half-life, calculated from the median tissue culture infectious dose (TCID50), saliva was protective of airborne murine coronavirus through a RH range recommended for conditioned indoor air (60% < RH < 40%; average half-life = 60 minutes). However, its average half-life doubled to 120 minutes when RH was maintained at 25%. Saliva microaerosol was dominated by carbohydrates, which presented hallmarks of vitrification without efflorescence at low RH. These results suggest that dehydrating carbohydrates can affect the infectious potential coronaviruses exhibit while airborne, significantly extending their persistence under the drier humidity conditions encountered indoors.

Transmission cluster of COVID-19 cases from Uruguay: emergence and spreading of a novel SARS-CoV-2 ORF6 deletion.

BACKGROUND: Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES: We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS: Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS: We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS: Our findings provide evidence for the origin and spread of deletion variants and emphasise indels' importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.

A deletion in SARS-CoV-2 ORF7 identified in COVID-19 outbreak in Uruguay.

The analysis of genetic diversity in SARS-CoV-2 is the focus of several studies, providing insights into how the virus emerged and evolves. Most common changes in SARS-CoV-2 are single or point nucleotide substitutions; meanwhile, insertions and deletions (indels) have been identified as a less frequent source of viral genetic variability. Here, we report the emergence of a 12-nucleotide deletion in ORF7a, resulting in a 4-amino acid in-frame deletion. The Δ12 variant was identified in viruses from patients of a single outbreak and represents the first report of this deletion in South American isolates. Phylogenetic analysis revealed that Δ12 strains belong to the lineage B.1.1 and clustered separated from the remaining Uruguayan strains. The ∆12 variant was detected in 14 patients of this outbreak by NGS sequencing and/or two rapid and economic methodologies: Sanger amplicon sequencing and capillary electrophoresis. The presence of strong molecular markers as the deletion described here are useful for tracking outbreaks and reveal a significant aspect of the SARS-CoV-2 evolution on the robustness of the virus to keep its functionality regardless loss of genetic material.

Genome Sequences of SARS-CoV-2 P.1 (Variant of Concern) and P.2 (Variant of Interest) Identified in Uruguay.

Two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants associated with increased transmission and immune evasion, P.1 and P.2, emerged in Brazil and spread throughout South America. Here, we report genomes corresponding to these variants that were recently detected in Uruguay. These P.1 and P.2 genomes share all substitutions that are characteristic of these variants.