SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here, we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 74-day time course. We show that changes in SARS-CoV-2 RNA concentrations follow symptom onset gathered by retrospective interview of patients but precedes clinical test results. In addition, we determine a nearly complete (98.5%) SARS-CoV-2 genome sequence from wastewater and use phylogenetic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for genotyping viral strains circulating in a community.

Temporal Detection and Phylogenetic Assessment of SARS-CoV-2 in Municipal Wastewater.

SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 52-day time course. We show that changes in SARS-CoV-2 RNA concentrations correlate with local COVID-19 epidemiological data (R2=0.9), though detection in wastewater trails symptom onset dates by 5-8 days. We determine a near complete (98.5%) SARS-CoV-2 genome sequence from the wastewater and use phylogenic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for high-resolution genotyping of the predominant strains circulating in a community.

https://www.medrxiv.org/content/medrxiv/early/2020/04/20/2020.04.15.20066746.full.pdf

Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named as 2019-nCoV) is responsible for the recent COVID-19 pandemic and polymerase chain reaction (PCR) is the current standard method for its diagnosis from patient samples. This study conducted a reassessment of published diagnostic PCR assays, including those recommended by the World Health Organization (WHO), through the evaluation of mismatches with publicly available viral sequences. An exhaustive evaluation of the sequence variability within the primer/probe target regions of the viral genome was performed using more than 17 000 viral sequences from around the world. The analysis showed the presence of mutations/mismatches in primer/probe binding regions of 7 assays out of 27 assays studied. A comprehensive bioinformatics approach for in silico inclusivity evaluation of PCR diagnostic assays of SARS-CoV-2 was validated using freely available software programs that can be applied to any diagnostic assay of choice. These findings provide potentially important information for clinicians, laboratory professionals and policy-makers.

https://royalsocietypublishing.org/doi/pdf/10.1098/rsos.200636

Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) causing coronavirus disease 2019 (COVID-19) has infected ten millions of people across the globe, and massive mutations in virus genome have occurred during the rapid spread of this novel coronavirus. Variance in protein sequence might lead to change in protein structure and interaction, then further affect the viral physiological characteristics, which could bring tremendous influence on the pandemic. In this study, we investigated 20 non-synonymous mutations in SARS-CoV-2 genome which incidence rates were all ≥1% as of September 1st, 2020, then modeled and analyzed the mutant protein structures. The results showed that four types of mutations caused dramatic changes in protein structures (RMSD ≥5.0 A), which were Q57H and G251V in open reading frames 3a (ORF3a), S194L and R203K/G204R in nucleocapsid (N). Next, we found that these mutations also affected the binding affinity of intraviral protein interactions. In addition, the hot spots within these docking mutant complexes were altered, among which the mutation Q57H was involved in both Orf3a-S and Orf3a-Orf8 protein interactions. Besides, these mutations were widely distributed all over the world, and their occurrences fluctuated as time went on. Notably, the incidences of R203K/G204R in N and Q57H in Orf3a were both over 50% in some countries. Overall, our findings suggest that SARS-CoV-2 mutations could change viral protein structure, binding affinity and hot spots of the interface, thereby might have impacts on SARS-CoV-2 transmission, diagnosis and treatment of COVID-19. This article is protected by copyright. All rights reserved.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jmv.26597

Effects of SARS-CoV-2 mutations on protein structures and intraviral protein-protein interactions.

Expansion of COVID-19 worldwide increases interest in unraveling genomic variations of novel SARS-CoV-2 virus. Metadata of 408,493 SARS-CoV-2 genomes submitted to GISAID database were analyzed with respect to genomic clades and their geographic, age, and gender distributions. Of the currently known SARS-CoV-2 clades, clade GR was the most prevalent worldwide followed by GV then GH. Chronological analysis revealed expansion in SARS-CoV-2 clades carrying D614G mutations with the predominance of the newest clade, GV, in the last three months. D614G clades prevail in countries with more COVID-19 cases. Of them, the clades GH and GR were more frequently recovered from severe or deceased COVID-19 cases. In contrast, G and GV clades showed a significantly higher prevalence among asymptomatic patients or those with mild disease. Metadata analysis showed higher (p < 0.05) prevalence of severe/deceased cases among males than females and predominance of GR clade in female patients. Furthermore, severe disease/death was more prevalent (p < 0.05) in elderly than in adults/children. Higher prevalence of the GV clade in children compared to other age groups was also evident. These findings uniquely provide a statistical evidence on the adaptation-driven evolution of SARS-CoV-2 leading to altered infectivity, virulence, and mortality.

/pdf/global-dynamics-of-sars-cov-2-clades-and-their-relation-to-kvpmnz3r5k.pdf

Global dynamics of SARS-CoV-2 clades and their relation to COVID-19 epidemiology.

The current pandemic caused by the new coronavirus is a worldwide public health concern. To aboard this emergency, and like never before, scientific groups around the world have been working in a fast and coordinated way to get the maximum of information about this virus when it has been almost 3 months since the first cases were detected in Wuhan province in China. The complete genome sequences of around 450 isolates are available, and studies about similarities and differences among them and with the close related viruses that caused similar epidemics in this century. In this work, we studied the complete genome of the first four cases of the new coronavirus disease in Chile, from patients who traveled to Europe and Southeast Asia. Our findings reveal at least two different viral variants entries to Chilean territory, coming from Europe and Asia. We also sub-classified the isolates into variants according to punctual mutations in the genome. Our work contributes to global information about transmission dynamics and the importance to take control measures to stop the spread of the infection.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/jmv.25797

Phylogenetic analysis of the first four SARS-CoV-2 cases in Chile.

The pandemic caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide public health concern. First confined in China and then disseminated widely across Europe and America, SARS-CoV-2 has impacted and moved the scientific community around the world to working in a fast and coordinated way to collect all possible information about this virus and generate new strategies and protocols to try to stop the infection. During March 2020, more than 16,000 full viral genomes have been shared in public databases that allow the construction of genetic landscapes for tracking and monitoring the viral advances over time and study the genomic variations present in geographic regions. In this work, we present the occurrence of genetic variants and lineages of SARS-CoV-2 in Chile during March to April 2020. Complete genome analysis of 141 viral samples from different regions of Chile revealed a predominance of variant D614G like in Europe and the USA and the major presence of lineage B.1. These findings could help take control measures due to the similarity of the viral variants present in Chile, compared with other countries, and monitor the dynamic change of virus variants in the country.

Geographical Distribution of Genetic Variants and Lineages of SARS-CoV-2 in Chile

Diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) cases is based on the count of real-time reverse transcription-plymerase chain reaction (RT-PCR) positive people. Viral load by real-time RT-PCR has been suggested as a biomarker of the SARS-CoV-2 infection. However, the association of viral load and severity of the disease is not yet resolved. Nasopharyngeal samples from 458 patients were tested by RT-PCR for SARS-CoV-2 diagnosis. Relative quantitation was made by the comparative threshold cycle (ΔΔCt ) formula between ORF1ab viral and RNase P housekeeping genes. Absolute viral load was calculate using a reference positive control. Most prevalent clinical signs were cough (75.8%), myalgia (66.7%), and fever (48.5%). Hypertension (18.2%), neurological diseases (15.1%), and asthma and hypothyroidism (12.1%) were most frequent comorbidities. Fever, either as an exclusive symptom or combined with others, was associated with high viral loads ( 2 - ∆ ∆ C t range, 35.65-155.16; 4.25-4.89 log10 RNA copies/test]). During the first week after onset of symptoms in mild patients up to 60 years-old was detected the peak of viral load. Children under 10 years old have a high viral load (313.84; 2.50) in the first 2 days postinfection with a sharp decline thereafter. Cases between 10 and 49 years old mostly showed low and moderate viral load during the first 2 days postinfection (range, 0.03 to 17.24; -1.50 to 1.24). Patients over 60 years old have high viral load up to the second week after the onset of symptoms (range, 25.32-155.42; 1.40-2.19), indicating the longer presence of the virus in them. These findings suggest the viral load in nasopharyngeal swabs would help to monitor the SARS-CoV-2 infection in mild coronavirus disease 2019 cases.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jmv.26761

Quantitative detection of SARS-CoV-2 RNA in nasopharyngeal samples from infected patients with mild disease.

Highlights Here we present the first human case of Influenza A H5N1 infection in Chile, and the fifth worldwide in 2023.The patient is a 53-year-old man who lives in the north region of Chile, near the seashore.The Chilean sample was subtyped in the clade 2.3.4.4b.

/pdf/the-first-case-of-human-infection-with-h5n1-avian-influenza-1d58ad5w.pdf

Carolina Tambley

Papers

Phylogenetic analysis of the first four SARS-CoV-2 cases in Chile.

Geographical Distribution of Genetic Variants and Lineages of SARS-CoV-2 in Chile

Quantitative detection of SARS-CoV-2 RNA in nasopharyngeal samples from infected patients with mild disease.

The first case of human infection with H5N1 avian influenza a virus in Chile.