Oral administration of obeldesivir protects nonhuman primates against Sudan ebolavirus.

Obeldesivir (ODV, GS-5245) is an orally administered prodrug of the parent nucleoside of remdesivir (RDV) and is presently in phase 3 trials for COVID-19 treatment. In this work, we show that ODV and its circulating parent nucleoside metabolite, GS-441524, have similar in vitro antiviral activity against filoviruses, including Marburg virus, Ebola virus, and Sudan virus (SUDV). We also report that once-daily oral ODV treatment of cynomolgus monkeys for 10 days beginning 24 hours after SUDV exposure confers 100% protection against lethal infection. Transcriptomics data show that ODV treatment delayed the onset of inflammation and correlated with antigen presentation and lymphocyte activation. Our results offer promise for the further development of ODV to control outbreaks of filovirus disease more rapidly.

Characterization of the Cynomolgus Macaque Model of Marburg Virus Disease and Assessment of Timing for Therapeutic Treatment Testing.

Marburg virus (MARV) causes severe disease and high mortality in humans. The objective of this study was to characterize disease manifestations and pathogenesis in cynomolgus macaques exposed to MARV. The results of this natural history study may be used to identify features of MARV disease useful in defining the ideal treatment initiation time for subsequent evaluations of investigational therapeutics using this model. Twelve cynomolgus macaques were exposed to a target dose of 1000 plaque-forming units MARV by the intramuscular route, and six control animals were mock-exposed. The primary endpoint of this study was survival to Day 28 post-inoculation (PI). Anesthesia events were minimized with the use of central venous catheters for periodic blood collection, and temperature and activity were continuously monitored by telemetry. All mock-exposed animals remained healthy for the duration of the study. All 12 MARV-exposed animals (100%) became infected, developed illness, and succumbed on Days 8-10 PI. On Day 4 PI, 11 of the 12 MARV-exposed animals had statistically significant temperature elevations over baseline. Clinically observable signs of MARV disease first appeared on Day 5 PI, when 6 of the 12 animals exhibited reduced responsiveness. Ultimately, systemic inflammation, coagulopathy, and direct cytopathic effects of MARV all contributed to multiorgan dysfunction, organ failure, and death or euthanasia of all MARV-exposed animals. Manifestations of MARV disease, including fever, systemic viremia, lymphocytolysis, coagulopathy, and hepatocellular damage, could be used as triggers for initiation of treatment in future therapeutic efficacy studies.

Use of the Xpert Breast Cancer STRAT4 for Biomarker Evaluation in Tissue Processed in a Developing Country.

OBJECTIVES: Breast cancer immunohistochemistry (IHC) biomarker testing is limited in low-resource settings, and an alternative solution is needed. A point-of-care mRNA STRAT4 breast cancer assay for ESR1, PGR, ERBB2, and MKi67, for use on the GeneXpert platform, has been recently validated on tissues from internationally accredited laboratories, showing excellent concordance with IHC. METHODS: We evaluated STRAT4/IHC ESR1/estrogen receptor (ER), ERBB2/human epidermal growth factor receptor 2 (HER2) concordance rates of 150 breast cancer tissues processed in Rwanda, with undocumented cold ischemic and fixation time. RESULTS: Assay fail/indeterminate rate was 2.6% for ESR1 and ERBB2. STRAT4 agreement with ER IHC was 92.5% to 93.3% and 97.8% for HER2, for standard (1x) and concentrated (4x) reagent-conserving protocols, respectively. Eleven of 12 discordant ER/ESR1 cases were ESR1- negative/IHC-positive. These had low expression of ER by IHC in mostly very small tumor areas tested (7/12; <25 mm2). In two of three discordant HER2 cases, the STRAT4-ERBB2 result correlated with the subsequent fluorescence in situ hybridization (FISH) result. STRAT4-ERBB2 results in 9 of 10 HER2-IHC equivocal cases were concordant with FISH. CONCLUSIONS: The STRAT4 assay is an alternative for providing quality-controlled breast cancer biomarker data in laboratories unable to provide quality and/or cost-efficient IHC services.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test.

Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.

Comparison of central laboratory assessments of ER, PR, HER2, and Ki67 by IHC/FISH and the corresponding mRNAs (ESR1, PGR, ERBB2, and MKi67) by RT-qPCR on an automated, broadly deployed diagnostic platform.

PURPOSE: The methods (IHC/FISH) typically used to assess ER, PR, HER2, and Ki67 in FFPE specimens from breast cancer patients are difficult to set up, perform, and standardize for use in low and middle-income countries. Use of an automated diagnostic platform (GeneXpert®) and assay (Xpert® Breast Cancer STRAT4) that employs RT-qPCR to quantitate ESR1, PGR, ERBB2, and MKi67 mRNAs from formalin-fixed, paraffin-embedded (FFPE) tissues facilitates analyses in less than 3 h. This study compares breast cancer biomarker analyses using an RT-qPCR-based platform with analyses using standard IHC and FISH for assessment of the same biomarkers. METHODS: FFPE tissue sections from 523 patients were sent to a College of American Pathologists-certified central reference laboratory to evaluate concordance between IHC/FISH and STRAT4 using the laboratory's standard of care methods. A subset of 155 FFPE specimens was tested for concordance with STRAT4 using different IHC antibodies and scoring methods. RESULTS: Concordance between STRAT4 and IHC was 97.8% for ESR1, 90.4% for PGR, 93.3% for ERBB2 (IHC/FISH for HER2), and 78.6% for MKi67. Receiver operating characteristic curve (ROC) area under the curve (AUC) values of 0.99, 0.95, 0.99, and 0.85 were generated for ESR1, PGR, ERBB2, and MKi67, respectively. Minor variabilities were observed depending on the IHC antibody comparator used. CONCLUSION: Evaluation of breast cancer biomarker status by STRAT4 was highly concordant with central IHC/FISH in this blinded, retrospectively analyzed collection of samples. STRAT4 may provide a means to cost-effectively generate standardized diagnostic results for breast cancer patients in low- and middle-income countries.

High concordance of a closed-system, RT-qPCR breast cancer assay for HER2 mRNA, compared to clinically determined immunohistochemistry, fluorescence in situ hybridization, and quantitative immunofluorescence.

Historically, mRNA measurements have been tested on several commercially available platforms, but none have gained broad acceptance for assessment of HER2. An mRNA measurement, as a continuous value, has the potential for use in adjudication of the equivocal category. Here we use a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay in a closed, single-use cartridge, automated system. Multiple cores (1 mm in diameter) were retrospectively collected from 80 formalin-fixed paraffin-embedded (FFPE) tissue blocks with invasive breast cancer seen by Yale Pathology Labs between 1998 and 2011. Tissue cores were processed with a FFPE lysis kit to create lysates that were tested with the automated RT-qPCR assay. Results for IHC and FISH were extracted from the pathology reports and quantitative immunofluorescence (QIF) for each case was measured as previously described. Quality control testing showed that the GX platform RT-qPCR shows no case to case cross contamination on material from routine histology practices. Concordance between RT-qPCR and IHC/FISH was 91.25% (sensitivity=0.87; specificity=0.94; PPV=0.89; NPV=0.92) using a pre-defined delta Ct cut-off (dCt≥-1) for HER2. Concordance (OPA) between RT-qPCR and QIF was 94% (sensitivity=0.90; specificity=0.96; PPV=0.93; NPV=0.94) using dCt≥-1 and a previously defined cut-point for positivity by QIF. In conclusion, the closed system RT-qPCR assay shows >90% concordance with the ASCO/CAP HER2 IHC/FISH scoring. Additionally, the RT-qPCR assay is highly concordant (94%) with the continuous variable HER2 QIF assay, and may better reflect the true continuum of HER2 receptor status in invasive breast cancer. These initial results suggest that fast, closed system molecular assays may have future value for the adjudication of the ASCO/CAP HER2 equivocal category or possibly routine usage in time constrained or low resource settings.

Persistent expression of hepatitis C virus non-structural proteins leads to increased autophagy and mitochondrial injury in human hepatoma cells.

HCV infection is a major cause of chronic liver disease and liver cancer in the United States. To address the pathogenesis caused by HCV infection, recent studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used human hepatoma cells constitutively replicating HCV RNA encoding either the full-length polyprotein or the non-structural proteins, or cells constitutively expressing the structural protein core, to model the state of persistent HCV infection and examined the combination of various HCV proteins in cellular pathogenesis. Increased reactive oxygen species (ROS) generation in the mitochondria, mitochondrial injury and degeneration, and increased lipid accumulation were common among all HCV protein-expressing cells regardless of whether they expressed the structural or non-structural proteins. Expression of the non-structural proteins also led to increased oxidative stress in the cytosol, membrane blebbing in the endoplasmic reticulum, and accumulation of autophagocytic vacuoles. Alterations of cellular redox state, on the other hand, significantly changed the level of autophagy, suggesting a direct link between oxidative stress and HCV-mediated activation of autophagy. With the wide-spread cytopathic effects, cells with the full-length HCV polyprotein showed a modest antioxidant response and exhibited a significant increase in population doubling time and a concomitant decrease in cyclin D1. In contrast, cells expressing the non-structural proteins were able to launch a vigorous antioxidant response with up-regulation of antioxidant enzymes. The population doubling time and cyclin D1 level were also comparable to that of control cells. Finally, the cytopathic effects of core protein appeared to focus on the mitochondria without remarkable disturbances in the cytosol.

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites.

The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1-S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2', R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell-cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2' cleavage site within S2 793-KPTKR-797 (S2'), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2' position allowed trypsin-independent cell-cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1-S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a critical proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2'), acting in concert with the S1-S2 cleavage site to mediate membrane fusion and virus infectivity.

Heparan sulfate is a selective attachment factor for the avian coronavirus infectious bronchitis virus Beaudette.

The avian coronavirus infectious bronchitis virus (IBV) strain Beaudette is an embryo-adapted virus that has extended species tropism in cell culture. In order to understand the acquired tropism of the Beaudette strain, we compared the S protein sequences of several IBV strains. The Beaudette strain was found to contain a putative heparan sulfate (HS)-binding site, indicating that the Beaudette virus may use HS as a selective receptor. To ascertain the requirements of cell-surface HS for Beaudette infectivity, we assayed for infectivity in the presence of soluble heparin as a competitor and determined infectivity in mutant cell lines with no HS or glycosaminoglycan expression. Our results indicate that HS plays a role as an attachment factor for IBV, working in concert with other factors like sialic acid to mediate virus binding to cells, and may explain in part the extended tropism of IBV Beaudette.

Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus.

BACKGROUND: Coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS), and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV). Feline aminopeptidase N (fAPN) serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV), canine coronavirus, transmissible gastroenteritis virus (TGEV), and human coronavirus 229E (HCoV-229E). A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047-2056. RESULTS: Here we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41), but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 (<0.01% efficiency), but this level of infection is not increased by fAPN expression. CONCLUSION: We conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation.

The avian coronavirus infectious bronchitis virus undergoes direct low-pH-dependent fusion activation during entry into host cells.

Coronaviruses are the causative agents of respiratory disease in humans and animals, including severe acute respiratory syndrome. Fusion of coronaviruses is generally thought to occur at neutral pH, although there is also evidence for a role of acidic endosomes during entry of a variety of coronaviruses. Therefore, the molecular basis of coronavirus fusion during entry into host cells remains incompletely defined. Here, we examined coronavirus-cell fusion and entry employing the avian coronavirus infectious bronchitis virus (IBV). Virus entry into cells was inhibited by acidotropic bases and by other inhibitors of pH-dependent endocytosis. We carried out fluorescence-dequenching fusion assays of R18-labeled virions and show that for IBV, coronavirus-cell fusion occurs in a low-pH-dependent manner, with a half-maximal rate of fusion occurring at pH 5.5. Fusion was reduced, but still occurred, at lower temperatures (20 degrees C). We observed no effect of inhibitors of endosomal proteases on the fusion event. These data are the first direct measure of virus-cell fusion for any coronavirus and demonstrate that the coronavirus IBV employs a direct, low-pH-dependent virus-cell fusion activation reaction. We further show that IBV was not inactivated, and fusion was unaffected, by prior exposure to pH 5.0 buffer. Virions also showed evidence of reversible conformational changes in their surface proteins, indicating that aspects of the fusion reaction may be reversible in nature.

Influenza virus entry and infection require host cell N-linked glycoprotein.

A widely held view of influenza virus infection is that the viral receptor consists of cell surface carbohydrate sialic acid, which can be present as glycoprotein or glycolipid. Here, we examined influenza virus entry and infection in Lec1 cells, a mutant CHO cell line deficient in terminal N-linked glycosylation caused by a mutation in the N-acetylglucosaminyltransferase I (GnT1) gene. We show that influenza virus cannot infect Lec1 cells, despite having full capacity to undergo virus binding and fusion. Lec1 cells also show no virus replication defect, and infection was restored in Lec1 cells expressing wild-type GnT1. Viruses were apparently arrested at the level of internalization from the plasma membrane and were not endocytosed. Lec1 cells were refractory to infection by several strains of influenza virus, including H1 and H3 strains of influenza A, as well as influenza B virus. Finally, cleavage of N-glycans from wild-type CHO cells markedly reduced infection by influenza virus. We suggest that influenza virus specifically requires N-linked glycoprotein for entry into cells, and that sialic acid, although acting as an efficient attachment factor, is not sufficient as an influenza virus receptor in vivo.

Modulation of the active complex assembly and turnover rate by protein-DNA interactions in Cre-LoxP recombination.

Cre promotes recombination at the 34 bp LoxP sequence. Substitution of a critical C-G base pair in LoxP with an A-T base pair, to give LoxAT, reduced Cre binding in vitro and abolished recombination in vivo [Hartung, M., and Kisters-Woike, B. (1998) J. Biol. Chem. 273, 22884-22891].We demonstrated that LoxAT can be recombined in vitro. However, Cre discriminates against this substrate both before and after DNA binding. The preference for LoxP over LoxAT is the result of reduced binding and a slower turnover rate, amplified by changes in cooperativity of complex assembly. With LoxAT, similar levels of substrate turnover required 2-2.5-fold higher protein-DNA concentrations compared to LoxP, but the sigmoidal behavior of the concentration dependence was more pronounced. Further, the Cre-LoxAT complexes reacted 4-5-fold more slowly. In the 2.3 A resolution Cre-LoxAT complex structure, the major groove Arg259-guanine interaction was disrupted, explaining the reduced binding. Overall structural shifts and mobility changes indicate more favorable interactions between subunits, providing a hypothesis for the reduced turnover rate. Concomitant with the displacement of Arg259 from the DNA, adjacent charged residues Glu262 and Glu266 shifted to form salt bridges with the Arg259 guanidinium moiety. Substitution of Glu262 and Glu266 with glutamine increased Cre complex assembly efficiency and reaction rates with both LoxAT and LoxP, but diminished Cre's ability to distinguish them. The increased rate of this variant suggests that DNA substrate binding and turnover are coupled. The improved efficiency, made at some expense of sequence discrimination, may be useful for enhancing recombination in vivo.

The order of strand exchanges in Cre-LoxP recombination and its basis suggested by the crystal structure of a Cre-LoxP Holliday junction complex.

Cre recombinase uses two pairs of sequential cleavage and religation reactions to exchange homologous DNA strands between 34 base-pair (bp) LoxP recognition sequences. In the oligomeric recombination complex, a switch between "cleaving" and "non-cleaving" subunit conformations regulates the number, order, and regio-specificity of the strand exchanges. However, the particular sequence of events has been in question. From analysis of strand composition of the Holliday junction (HJ) intermediate, we determined that Cre initiates recombination of LoxP by cleaving the upper strand on the left arm. Cre preferred to react with the left arm of a LoxP suicide substrate, but at a similar rate to the right arm, indicating that the first strand to be exchanged is selected prior to cleavage. We propose that during complex assembly the cleaving subunit preferentially associates with the LoxP left arm, directing the first strand exchange to that side. In addition, this biased assembly would enforce productive orientation of LoxP sites in the recombination synapses. A novel Cre-HJ complex structure in which LoxP was oriented with the left arm bound by the cleaving Cre subunit suggested a physical basis for the strand exchange order. Lys86 and Lys201 interact with the left arm scissile adenine base differently than in structures that have a scissile guanine. These interactions are associated with positioning the 198-208 loop, a structural component of the conformational switch, in a configuration that is specific to the cleaving conformation. Our results suggest that strand exchange order and site alignment are regulated by an "induced fit" mechanism in which the cleaving conformation is selectively stabilized through protein-DNA interactions with the scissile base on the strand that is cleaved first.