SARS-CoV-2 innate effector associations and viral load in early nasopharyngeal infection.

COVID-19 causes severe disease with poor outcomes. We tested the hypothesis that early SARS-CoV-2 viral infection disrupts innate immune responses. These changes may be important for understanding subsequent clinical outcomes. We obtained residual nasopharyngeal swab samples from individuals who requested COVID-19 testing for symptoms at drive-through COVID-19 clinical testing sites operated by the University of Utah. We applied multiplex immunoassays, real-time polymerase chain reaction assays and quantitative proteomics to 20 virus-positive and 20 virus-negative samples. ACE-2 transcripts increased with infection (OR =17.4, 95% CI [CI] =4.78-63.8) and increasing viral N1 protein transcript load (OR =1.16, CI =1.10-1.23). Transcripts for two interferons (IFN) were elevated, IFN-λ1 (OR =71, CI =7.07-713) and IFN-λ2 (OR =40.2, CI =3.86-419), and closely associated with viral N1 transcripts (OR =1.35, CI =1.23-1.49 and OR =1.33 CI =1.20-1.47, respectively). Only transcripts for IP-10 were increased among systemic inflammatory cytokines that we examined (OR =131, CI =1.01-2620). We found widespread discrepancies between transcription and translation. IFN proteins were unchanged or decreased in infected samples (IFN-γ OR =0.90 CI =0.33-0.79, IFN-λ2,3 OR =0.60 CI =0.48-0.74) suggesting viral-induced shut-off of host antiviral protein responses. However, proteins for IP-10 (OR =3.74 CI =2.07-6.77) and several interferon-stimulated genes (ISG) increased with viral load (BST-1 OR =25.1, CI =3.33-188; IFIT1 OR =19.5, CI =4.25-89.2; IFIT3 OR =245, CI =15-4020; MX-1 OR =3.33, CI =1.44-7.70). Older age was associated with substantial modifications of some effects. Ambulatory symptomatic patients had an innate immune response with SARS-CoV-2 infection characterized by elevated IFN, proinflammatory cytokine and ISG transcripts, but there is evidence of a viral-induced host shut-off of antiviral responses. Our findings may characterize the disrupted immune landscape common in patients with early disease.

SARS-CoV-2 Innate Effector Associations and Viral Load in Early Nasopharyngeal Infection.

To examine innate immune responses in early SARS-CoV-2 infection that may change clinical outcomes, we compared nasopharyngeal swab data from 20 virus-positive and 20 virus-negative individuals. Multiple innate immune-related and ACE-2 transcripts increased with infection and were strongly associated with increasing viral load. We found widespread discrepancies between transcription and translation. Interferon proteins were unchanged or decreased in infected samples suggesting virally-induced shut-off of host anti-viral protein responses. However, IP-10 and several interferon-stimulated gene proteins increased with viral load. Older age was associated with modifications of some effects. Our findings may characterize the disrupted immune landscape of early disease.

Clinical evaluation of the BioFire® Respiratory Panel 2.1 and detection of SARS-CoV-2.

We evaluated the performance of the BioFire® Respiratory Panel 2.1 (RP2.1) in the detection of SARS CoV-2 in comparison against three other SARS CoV-2 EUA assays. In these studies, the RP2.1 panel had 98 % positive percent agreement (48/49) and 100 % negative percent agreement (49/49). Since 30 % of nasopharyngeal swab specimens have a SARS CoV-2 Ct >30 and thus detection of virus in low titers is clinically relevant, a sample with a high titer was diluted and each 10 fold dilution was tested in triplicate and compared against 6 other EUA approved SARS CoV-2 assays. These data suggested that the BioFire® RP2.1 panel, along with four other SARS CoV-2 assays (Roche cobas, Cepheid Xpert Xpress, BioFire® Defense COVID19, and NECoV19), consistently detected viral RNA at the 10-7 dilution. Overall, these studies suggest that the BioFire® RP2.1 assay can be used to detect acute cases of SARS CoV2 in addition to patients with low viral titer later in disease presentation.

Clinical Performance of SurePath™ Preservative Compared to PreservCyt® with Cobas® and Hybrid Capture® 2 HPV Tests in a Colposcopy Population.

BACKGROUND: Until recently, no HPV test had been US FDA-approved for SurePath preservative. Clinical performance remains incompletely understood. The clinical performances of the Cobas HPV Test (Cobas) and Hybrid Capture 2 High-Risk HPV DNA Test (HC2) with PreservCyt and SurePath preservatives were compared. METHODS: Cervical cytology samples were collected in both preservatives in random order from women age 21+ (n = 244) referred for colposcopy. Before cytology processing and pelleting, SurePath samples were tested by the Cobas test with and without buffered SDS heat pretreatment. SurePath pellets were tested by the HC2 test and by the Cobas test (with pretreatment). Performance characteristics were calculated in relation to cases of cervical in-traepithelial neoplasia grade 2 or higher (CIN2+) as the clinical target outcome. All HPV-positive samples were also genotyped with the Linear Array test. RESULTS: CIN2+ was detected in 42 patients (17.2%). For both HPV tests, there was a trend towards higher positivity and sensitivity for SurePath compared to PreservCyt preservative. The Cobas test had higher sensitivity than HC2 and the HC2 test had higher specificity than Cobas. Pretreated SurePath samples produced results similar to untreated ones, despite a two-fold dilution during pretreatment [sensitivity %: 95.1 (82.2 - 99.2) vs. 94.3 (79.5 - 99.0); specificity %: 33.0 (26.6 - 40.1) vs. 33.0 (26.4 - 40.3)]. CONCLUSIONS: There was good agreement between the preservatives and HPV tests in detecting HPV and between the Cobas and Linear Array tests for genotyping HR-HPV. These trends were not statistically significant due to the limited number of CIN2+ cases. However, these data may help in evaluations of preservative selection for colposcopy samples. Pre-treatment for Cobas testing eliminated invalid results due to clots. The Cobas test has been FDA-approved for use with heat pretreated SurePath samples.

Human Bocavirus Capsid Messenger RNA Detection in Children With Pneumonia.

Background: The role of human bocavirus (HBoV) in respiratory illness is uncertain. HBoV genomic DNA is frequently detected in both ill and healthy children. We hypothesized that spliced viral capsid messenger RNA (mRNA) produced during active replication might be a better marker for acute infection. Methods: As part of the Etiology of Pneumonia in the Community (EPIC) study, children aged <18 years who were hospitalized with community-acquired pneumonia (CAP) and children asymptomatic at the time of elective outpatient surgery (controls) were enrolled. Nasopharyngeal/oropharyngeal specimens were tested for HBoV mRNA and genomic DNA by quantitative polymerase chain reaction. Results: HBoV DNA was detected in 10.4% of 1295 patients with CAP and 7.5% of 721 controls (odds ratio [OR], 1.4 [95% confidence interval {CI}, 1.0-2.0]); HBoV mRNA was detected in 2.1% and 0.4%, respectively (OR, 5.1 [95% CI, 1.6-26]). When adjusted for age, enrollment month, and detection of other respiratory viruses, HBoV mRNA detection (adjusted OR, 7.6 [95% CI, 1.5-38.4]) but not DNA (adjusted OR, 1.2 [95% CI, .6-2.4]) was associated with CAP. Among children with no other pathogens detected, HBoV mRNA (OR, 9.6 [95% CI, 1.9-82]) was strongly associated with CAP. Conclusions: Detection of HBoV mRNA but not DNA was associated with CAP, supporting a pathogenic role for HBoV in CAP. HBoV mRNA could be a useful target for diagnostic testing.

Viral Pathogen Detection by Metagenomics and Pan-Viral Group Polymerase Chain Reaction in Children With Pneumonia Lacking Identifiable Etiology.

Background: Community-acquired pneumonia (CAP) is a leading cause of pediatric hospitalization. Pathogen identification fails in approximately 20% of children but is critical for optimal treatment and prevention of hospital-acquired infections. We used two broad-spectrum detection strategies to identify pathogens in test-negative children with CAP and asymptomatic controls. Methods: Nasopharyngeal/oropharyngeal (NP/OP) swabs from 70 children <5 years with CAP of unknown etiology and 90 asymptomatic controls were tested by next-generation sequencing (RNA-seq) and pan viral group (PVG) PCR for 19 viral families. Association of viruses with CAP was assessed by adjusted odds ratios (aOR) and 95% confidence intervals controlling for season and age group. Results: RNA-seq/PVG PCR detected previously missed, putative pathogens in 34% of patients. Putative viral pathogens included human parainfluenza virus 4 (aOR 9.3, P = .12), human bocavirus (aOR 9.1, P < .01), Coxsackieviruses (aOR 5.1, P = .09), rhinovirus A (aOR 3.5, P = .34), and rhinovirus C (aOR 2.9, P = .57). RNA-seq was more sensitive for RNA viruses whereas PVG PCR detected more DNA viruses. Conclusions: RNA-seq and PVG PCR identified additional viruses, some known to be pathogenic, in NP/OP specimens from one-third of children hospitalized with CAP without a previously identified etiology. Both broad-range methods could be useful tools in future epidemiologic and diagnostic studies.

Development of a real-time PCR assay for the direct detection of Candida species causing Vulvovaginal candidiasis.

Identification of Candida species by traditional methods can be time-consuming and have limited analytical sensitivity. We developed a multiplex real-time PCR assay for detection and differentiation of Candida species causing vulvovaginal candidiasis (VVC). Overall, this PCR assay is a powerful diagnostic tool offering superior accuracy, sensitivity, and specificity.

Taxonomer: an interactive metagenomics analysis portal for universal pathogen detection and host mRNA expression profiling.

BACKGROUND: High-throughput sequencing enables unbiased profiling of microbial communities, universal pathogen detection, and host response to infectious diseases. However, computation times and algorithmic inaccuracies have hindered adoption. RESULTS: We present Taxonomer, an ultrafast, web-tool for comprehensive metagenomics data analysis and interactive results visualization. Taxonomer is unique in providing integrated nucleotide and protein-based classification and simultaneous host messenger RNA (mRNA) transcript profiling. Using real-world case-studies, we show that Taxonomer detects previously unrecognized infections and reveals antiviral host mRNA expression profiles. To facilitate data-sharing across geographic distances in outbreak settings, Taxonomer is publicly available through a web-based user interface. CONCLUSIONS: Taxonomer enables rapid, accurate, and interactive analyses of metagenomics data on personal computers and mobile devices.

Cervical Cytology Specimen Stability in Surepath Preservative and Analytical Sensitivity for HPV Testing with the cobas and Hybrid Capture 2 Tests.

None of the commercial HPV tests are U.S. FDA-approved for testing of cervical cytology specimens in SurePath preservative. Still, ~30% of HPV testing is performed on specimens in this formalin-containing preservative. Formalin-induced DNA fragmentation and cross-linking may interfere with HPV detection. We evaluated analytical sensitivity and specimen stability of the cobas 4800 HPV (Roche) and Hybrid Capture 2 HPV (HC2, Qiagen) tests with residual cervical cytology samples in SurePath preservative available within 1 week of collection. Cobas testing was performed with and without heating samples at 120°C for 20 min diluted 1:1 in an alkaline environment (pretreatment) to revert DNA crosslinking. Stability was tested after 2 weeks of storage at ambient temperature followed by ≤10 weeks at 4°C. Analytical sensitivity and positivity rates (HC2, 18%; cobas pretreated, 46%; cobas untreated, 47%) were greater for cobas than HC2 (n = 682). After 6 weeks of storage, mean HC2 ratios were lower (mean 0.9, SD 6.3) but high variability limited statistical power to detect trends. Cobas threshold cycles (Ct's) increased in untreated (mean 2.1) but not pretreated samples (mean 0.3; n = 110; p≤0.0001). Overall, cobas had greater analytical sensitivity for samples in SurePath preservative. Although pretreatment introduced a manual sample transfer step and 30 min of incubation times, it improved stability without negatively affecting analytical sensitivity. While awaiting results of large trials to evaluate the clinical performance of cobas, the addition of the pretreatment step may improve the detection of HPV, especially after prolonged sample storage.

Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel.

Current infectious disease molecular tests are largely pathogen specific, requiring test selection based on the patient's symptoms. For many syndromes caused by a large number of viral, bacterial, or fungal pathogens, such as respiratory tract infections, this necessitates large panels of tests and has limited yield. In contrast, next-generation sequencing-based metagenomics can be used for unbiased detection of any expected or unexpected pathogen. However, barriers for its diagnostic implementation include incomplete understanding of analytical performance and complexity of sequence data analysis. We compared detection of known respiratory virus-positive (n= 42) and unselected (n= 67) pediatric nasopharyngeal swabs using an RNA sequencing (RNA-seq)-based metagenomics approach and Taxonomer, an ultrarapid, interactive, web-based metagenomics data analysis tool, with an FDA-cleared respiratory virus panel (RVP; GenMark eSensor). Untargeted metagenomics detected 86% of known respiratory virus infections, and additional PCR testing confirmed RVP results for only 2 (33%) of the discordant samples. In unselected samples, untargeted metagenomics had excellent agreement with the RVP (93%). In addition, untargeted metagenomics detected an additional 12 viruses that were either not targeted by the RVP or missed due to highly divergent genome sequences. Normalized viral read counts for untargeted metagenomics correlated with viral burden determined by quantitative PCR and showed high intrarun and interrun reproducibility. Partial or full-length viral genome sequences were generated in 86% of RNA-seq-positive samples, allowing assessment of antiviral resistance, strain-level typing, and phylogenetic relatedness. Overall, untargeted metagenomics had high agreement with a sensitive RVP, detected viruses not targeted by the RVP, and yielded epidemiologically and clinically valuable sequence information.

High-risk HPV detection and genotyping by APTIMA HPV using cervical samples.

High-risk human papillomavirus (HPV) detection and genotyping is critical for cervical cancer screening. Testing of 967 cervical cytology specimens in PreservCyt preservative revealed similar positivity rates for HC2 (13.8%) and APTIMA HPV (AHPV) tests (13.5%, p=0.89) and high overall agreement (94.6%, κ=0.77). A trend towards higher HPV16 positivity rates by the Cobas HPV test (23.0%, 26/113) compared to the AHPV genotyping assay (19.5%, 22/113; p=0.125) was noted. No cross-contamination was detected with AHPV in a challenge experiment.

Simultaneous titration and phenotypic antiviral drug susceptibility testing for herpes simplex virus 1 and 2.

BACKGROUND: Most herpes simplex virus (HSV) isolates from treatment-naïve patients are susceptible to antivirals. However, prolonged antiviral therapy can select for drug-resistant strains, especially in immunocompromised patients. Standard phenotypic methods for antiviral resistance testing are labor and time-intense and molecular resistance determinants are insufficiently understood for routine diagnostic use of genotypic resistance testing. OBJECTIVE: To enable rapid, scalable antiviral susceptibility testing and minimize viral passage, we developed a 7-day, 96-well assay for simultaneous HSV 1/2 titration and phenotypic resistance testing for acyclovir and foscarnet. STUDY DESIGN: The assay was optimized and validated by testing clinical isolates and laboratory strains (n=39) with known IC50 for acyclovir (23 resistant) and foscarnet (1 resistant) based on plaque reduction or dye-uptake assays. A chemiluminescent detection reagent is used for quantification of cytopathic effect instead of plaque counting or measuring dye-uptake. Drug concentrations inhibiting 50% of chemiluminescent signal reduction (IC50) were determined concurrently at each of three virus dilutions. RESULTS: Results agree for 92.3% (acyclovir) and 100% (foscarnet) of isolates. For all three discordant samples, results of reference testing by plaque reduction agreed with the chemiluminescent assay. Reproducibility studies showed 100% qualitative agreement and 3-37% coefficient of variation based on IC50. CONCLUSIONS: Chemiluminescence detection as a surrogate for cellular viability with an automated plate reader provides improved throughput and workflow, as well as high accuracy and reproducibility for antiviral drug susceptibility testing.

Anti-HIV-1 activity of Trim 37.

Trim 5α was the first member of the tripartite motif (TRIM) family of proteins that was identified to potently restrict human immunodeficiency virus type 1 (HIV-1) replication. The breadth of antiretroviral activity of TRIM family members is an active area of investigation. In this study, we demonstrate that human Trim 37 possesses anti-HIV-1 activity. This antiretroviral activity and the manner in which it was displayed were implicated by (1) decreased viral replication upon Trim 37 transient overexpression in virus-producing cells, (2) correlation of the reduction of viral infectivity with Trim 37 virion incorporation, (3) increased HIV-1 replication during siRNA depletion of Trim 37 expression, and (4) reduction in viral DNA synthesis upon Trim 37 transient overexpression. Our findings provide the first demonstration, to our knowledge, of the potent antiviral activity of human Trim 37, and implicate an antiviral mechanism whereby Trim 37 interferes with viral DNA synthesis.

Sequencing-based genotyping of mixed human papillomavirus infections by use of RipSeq software.

Sequencing-based pathogen identification directly from clinical specimens requires time-consuming interpretation, especially with mixed chromatograms when multiple microorganisms are detected. We assessed RipSeq Mixed software for human papillomavirus (HPV) genotyping by comparison to the linear array HPV genotyping assay. RipSeq Mixed provided rapid, sequencing-based HPV typing for single-type infections and coinfections with 2 types.

Characterization of the cellular and antitumor effects of MPI-0479605, a small-molecule inhibitor of the mitotic kinase Mps1.

Mps1 is a dual specificity protein kinase that is essential for the bipolar attachment of chromosomes to the mitotic spindle and for maintaining the spindle assembly checkpoint until all chromosomes are properly attached. Mps1 is expressed at high levels during mitosis and is abundantly expressed in cancer cells. Disruption of Mps1 function induces aneuploidy and cell death. We report the identification of MPI-0479605, a potent and selective ATP competitive inhibitor of Mps1. Cells treated with MPI-0479605 undergo aberrant mitosis, resulting in aneuploidy and formation of micronuclei. In cells with wild-type p53, this promotes the induction of a postmitotic checkpoint characterized by the ATM- and RAD3-related-dependent activation of the p53-p21 pathway. In both wild-type and p53 mutant cells lines, there is a growth arrest and inhibition of DNA synthesis. Subsequently, cells undergo mitotic catastrophe and/or an apoptotic response. In xenograft models, MPI-0479605 inhibits tumor growth, suggesting that drugs targeting Mps1 may have utility as novel cancer therapeutics.

Simple but highly effective three-dimensional chemical-feature-based pharmacophore model for diketo acid derivatives as hepatitis C virus RNA-dependent RNA polymerase inhibitors.

A molecular modeling strategy using aryl diketo acid (ADK) derivatives recently reported in the literature as hepatitis C virus (HCV) polymerase inhibitors was designed. A 3D chemical-feature-based pharmacophore model was developed using Catalyst software, which produced 10 pharmacophore hypotheses. The top-ranked one (Hypo 1), characterized by a high correlation coefficient (r = 0.965), consisted of two hydrogen bond acceptors, one negative ionizable moiety, and two hydrophobic aromatics. This model was used to predict the anti-RNA-dependent RNA polymerase (anti-RdRp) activity of 6-(1-arylmethylpyrrol-2-yl)-1,4-dioxo-5-hexenoic acids and other ADK derivatives previously synthesized in our laboratories as HIV-1 integrase inhibitors. Furthermore, the experimental IC50 values of 9 compounds, tested in vitro against recombinant HCV polymerase, were compared with the corresponding values predicted using Hypo1. A good agreement between experimental and simulated data was obtained. The results demonstrate that the hypothesis derived in this study can be considered to be a useful tool in designing new leads based on ADK scaffolds as HCV RdRp inhibitors.

Hepatitis C virus, ER stress, and oxidative stress.

Hepatitis C virus (HCV) replication is associated with the endoplasmic reticulum (ER), where the virus causes stress. Cells cope with ER stress by activating an adaptive program called the unfolded protein response (UPR), which alleviates this stress by stimulating protein folding and degradation in the ER and down-regulating overall protein synthesis. Recent work suggests that HCV also alters ER calcium homeostasis, inducing oxidative stress. Future progress in understanding the control that HCV exerts over the ER will provide insight into viral strategies for pathogenesis and persistence in chronically infected patients.

Functional group recognition at the aminoacylation and editing sites of E. coli valyl-tRNA synthetase.

To correct misactivation and misacylation errors, Escherichia coli valyl-tRNA synthetase (ValRS) catalyzes a tRNA(Val)-dependent editing reaction at a site distinct from its aminoacylation site. Here we examined the effects of replacing the conserved 3'-adenosine of tRNA(Val) with nucleoside analogs, to identify structural elements of the 3'-terminal nucleoside necessary for tRNA function at the aminoacylation and editing sites of ValRS. The results show that the exocyclic amino group (N6) is not essential: purine riboside-substituted tRNA(Val) is active in aminoacylation and in stimulating editing. Presence of an O6 substituent (guanosine, inosine, xanthosine) interferes with aminoacylation as well as posttransfer and total editing (pre- plus posttransfer editing). Because ValRS does not recognize substituents at the 6-position, these results suggest that an unprotonated N1, capable of acting as an H-bond acceptor, is an essential determinant for both the aminoacylation and editing reactions. Substituents at the 2-position of the purine ring, either a 2-amino group (2-aminopurine, 2,6-diaminopurine, guanosine, and 7-deazaguanosine) or a 2-keto group (xanthosine, isoguanosine), strongly inhibit both aminoacylation and editing. Although aminoacylation by ValRS is at the 2'-OH, substitution of the 3'-terminal adenosine of tRNA(Val) with 3'-deoxyadenosine reduces the efficiency of valine acceptance and of posttransfer editing, demonstrating that the 3'-terminal hydroxyl group contributes to tRNA recognition at both the aminoacylation and editing sites. Our results show a strong correlation between the amino acid accepting activity of tRNA and its ability to stimulate editing, suggesting misacylated tRNA is a transient intermediate in the editing reaction, and editing by ValRS requires a posttransfer step.

Hepatitis C virus suppresses the IRE1-XBP1 pathway of the unfolded protein response.

Hepatitis C virus (HCV) gene expression disrupts normal endoplasmic reticulum (ER) functions and induces ER stress. ER stress results from the accumulation of unfolded or misfolded proteins in the ER; cells can alleviate this stress by degrading or refolding these proteins. The IRE1-XBP1 pathway directs both protein refolding and degradation in response to ER stress. Like IRE1-XBP1, other branches of the ER stress response mediate protein refolding. However, IRE1-XBP1 can also specifically activate protein degradation. We show here that XBP1 expression is elevated in cells carrying HCV subgenomic replicons, but XBP1 trans-activating activity is repressed. This prevents the IRE1-XBP1 transcriptional induction of EDEM (ER degradation-enhancing alpha-mannosidase-like protein). The mRNA expression of EDEM is required for the degradation of misfolded proteins. Consequently, misfolded proteins are stable in cells expressing HCV replicons. HCV may suppress the IRE1-XBP1 pathway to stimulate the synthesis of its viral proteins. IRE1alpha-null MEFs, a cell line with a defective IRE1-XBP1 pathway, show elevated levels of HCV IRES-mediated translation. Therefore, HCV may suppress the IRE1-XBP1 pathway to not only promote HCV expression but also to contribute to the persistence of the virus in infected hepatocytes.