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1.
Viruses ; 14(2)2022 02 17.
Article in English | MEDLINE | ID: covidwho-1704412

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have spilled over from humans to companion and wild animals since the inception of the global COVID-19 pandemic. However, whole genome sequencing data of the viral genomes that infect non-human animal species have been scant. Here, we detected and sequenced a SARS-CoV-2 delta variant (AY.3) in fecal samples from an 11-year-old domestic house cat previously exposed to an owner who tested positive for SARS-CoV-2. Molecular testing of two fecal samples collected 7 days apart yielded relatively high levels of viral RNA. Sequencing of the feline-derived viral genomes showed the two to be identical, and differing by between 4 and 14 single nucleotide polymorphisms in pairwise comparisons to human-derived lineage AY.3 sequences collected in the same geographic area and time period. However, several mutations unique to the feline samples reveal their divergence from this cohort on phylogenetic analysis. These results demonstrate continued spillover infections of emerging SARS-CoV-2 variants that threaten human and animal health, as well as highlight the importance of collecting fecal samples when testing for SARS-CoV-2 in animals. To the authors' knowledge, this is the first published case of a SARS-CoV-2 delta variant in a domestic cat in the United States.


Subject(s)
COVID-19/veterinary , Feces/virology , Pets/virology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Animals , COVID-19/transmission , COVID-19/virology , Cats , Female , Genome, Viral/genetics , Humans , Phylogeny , RNA, Viral/genetics , SARS-CoV-2/classification , United States , Whole Genome Sequencing
2.
mBio ; : e0378821, 2022 Feb 08.
Article in English | MEDLINE | ID: covidwho-1673352

ABSTRACT

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of1.25-3.18). This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. IMPORTANCE SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here, we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in southeastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a 3-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

3.
J Infect Dis ; 2022 Feb 02.
Article in English | MEDLINE | ID: covidwho-1672211

ABSTRACT

Some risk factors for severe COVID-19 have been identified, including age, race, and obesity. However, 20-50% of severe cases occur in the absence of these factors. Cytomegalovirus (CMV) is a herpes virus that infects ~50% of all individuals worldwide and is one of the most significant non-genetic determinants of immune system. We hypothesized that latent CMV infection might influence the severity of COVID-19. Our analyses demonstrate that CMV seropositivity associates with more than twice the risk of hospitalization due to SARS-CoV-2 infection. Immune profiling of blood and CMV DNA qPCR in a subset of patients for whom respiratory tract samples were available revealed altered T cell activation profiles in absence of extensive CMV replication in the upper respiratory tract. These data suggest a potential role for CMV-driven immune perturbations in affecting the outcome of SARS-CoV-2 infection and may have implications for the discrepancies in COVID-19 severity between different human populations.

4.
Journal of biomolecular techniques : JBT ; 32(3):98-101, 2021.
Article in English | EuropePMC | ID: covidwho-1619369

ABSTRACT

Over the course of the coronavirus disease 2019 (COVID-19) pandemic, several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic variants of concern have appeared and spread throughout the world. Detection and identification of these variants are important to understanding and controlling their rapid spread. Current detection methods for a particularly concerning variant, B.1.1.7, require expensive quantitative PCR machines and depend on the absence of a signal rather than a positive indicator of variant presence. Here we report an assay using a pair of molecular beacons combined with reverse transcription loop mediated amplification to allow isothermal amplification from saliva to specifically detect B.1.1.7 and other variants that contain a characteristic deletion in the gene encoding the viral spike protein. This assay is specific and affordable and allows multiplexing with other SARS-CoV-2 loop-mediated amplification primer sets.

5.
Genome Biol ; 22(1): 169, 2021 06 03.
Article in English | MEDLINE | ID: covidwho-1388811

ABSTRACT

BACKGROUND: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. RESULTS: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. CONCLUSIONS: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.


Subject(s)
COVID-19/diagnosis , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , COVID-19 Testing , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Nucleic Acid Probes/genetics , SARS-CoV-2/genetics , Saliva/virology , Sensitivity and Specificity
6.
mBio ; 12(4): e0177721, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1360545

ABSTRACT

Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Here, we investigated the respiratory tract microbiome in coronavirus disease 2019 (COVID-19) and its relationship to disease severity, systemic immunologic features, and outcomes. We examined 507 oropharyngeal, nasopharyngeal, and endotracheal samples from 83 hospitalized COVID-19 patients as well as non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, and the commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR. We found that COVID-19 patients had upper respiratory microbiome dysbiosis and greater change over time than critically ill patients without COVID-19. Oropharyngeal microbiome diversity at the first time point correlated inversely with disease severity during hospitalization. Microbiome composition was also associated with systemic immune parameters in blood, as measured by lymphocyte/neutrophil ratios and immune profiling of peripheral blood mononuclear cells. Intubated patients showed patient-specific lung microbiome communities that were frequently highly dynamic, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Thus, the respiratory tract microbiome and commensal viruses are disturbed in COVID-19 and correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, its possible use as biomarkers, and the role of bacterial and viral taxa identified here in COVID-19 pathogenesis. IMPORTANCE COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Early airway samples tracked with the level of COVID-19 illness reached during hospitalization, and the airway microbiome also correlated with immune parameters in blood. These findings raise questions about the mechanisms linking SARS-CoV-2 infection and other microbial inhabitants of the airway, including whether the microbiome might regulate severity of COVID-19 disease and/or whether early microbiome features might serve as biomarkers to discriminate disease severity.


Subject(s)
Bacteria/classification , Dysbiosis/microbiology , Lung/microbiology , Nasopharynx/microbiology , Oropharynx/microbiology , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Anelloviridae/classification , Anelloviridae/genetics , Anelloviridae/isolation & purification , Bacteria/genetics , Bacteria/isolation & purification , COVID-19/pathology , Female , Humans , Lymphocyte Count , Male , Microbiota , Middle Aged , RNA, Ribosomal, 16S/genetics , Severity of Illness Index
7.
Clin Chem ; 68(1): 230-239, 2021 12 30.
Article in English | MEDLINE | ID: covidwho-1354284

ABSTRACT

BACKGROUND: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients. RESULTS: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.


Subject(s)
Antigens, Viral/analysis , COVID-19 Testing/methods , COVID-19 , Smartphone , COVID-19/diagnosis , Humans , Machine Learning , SARS-CoV-2 , Sensitivity and Specificity
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