Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Chronic hepatitis B virus (HBV) infection can result in severe liver disease with eventual progression to cirrhosis and hepatocellular carcinoma. 1 Roughly 5% of the world' s population (over 350 million persons) are chronically infected with HBV. 2 Although interferon alfa remains the only licensed drug for the treatment of chronic HBV infection, the overall response rate to this immunotherapy is less than 40%. 3 Therefore, other effective antiviral therapies for patients with HBV infection are needed.Lamivudine [(-)2Ј-deoxy-3Ј-thiacytidine, 3TC] is a member of a class of antiviral nucleoside analogs that inhibit hepadnavirus replication specifically by terminating viral DNA synthesis. 4 Lamivudine is currently being evaluated in Phase III clinical trials for the treatment of both patients chronically infected with HBV and patients with HBV reinfection of an allograft after orthotopic liver transplantation.In Phase II trials, 5-7 lamivudine treatment was shown to rapidly reduce serum HBV DNA to levels below the detection limit of standard commercial assays, and to be well tolerated with no major toxicities. However, as with other antivirals, 7-11 resistance to lamivudine therapy can emerge in some patients. Recent investigations have reported the development of lamivudine-resistant HBV in six orthotopic liver transplantation patients on therapy. [12][13][14] In each patient, sequence analysis of serum HBV DNA revealed the presence of specific mutations in the tyrosine, methionine, aspartate, aspartate (YMDD) amino acid motif of the viral polymerase. This YMDD motif is a conserved domain of all reverse transcriptases (RT) and is required for polymerization activity. However, various other amino acid changes in the polymerase were also described in these reports.To determine the significance of various mutations in the development of lamivudine-resistant HBV, a more comprehensive study was undertaken. In this study, DNA sequences were determined from HBV isolates from 20 patients experiencing breakthrough HBV reactivation while on lamivudine therapy. From this larger series of 20 clinical HBV isolates, the database of lamivudine-resistant HBV sequences was expanded to confirm viral DNA mutations associated with lamivudine resistance in vivo. To explore the biological significance of the key observed mutations, putative resis-

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.

Molecular diagnosis of COVID-19 primarily relies on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RT-PCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity. We highlight the main issues surrounding molecular diagnosis of COVID-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. We discuss critical research needs, such as improvements in RT-PCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Improved assays are also needed for environmental surveillance or wastewater-based epidemiology, which gauges infection on the community level through analyses of viral components in the community's wastewater. Public health surveillance benefits from large-scale analyses of antibodies in serum, although the current serological tests do not quantify neutralizing antibodies. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of COVID-19 are valuable for better preparedness in response to other infectious diseases.

ells expressing ACE2 are potential targets of SARS-CoV-2 infection 1,2. Studies based on single-cell RNA sequencing (scRNA-seq) of lung cells have identified type II pneumocytes, ciliated cells and transient secretory cells as the main types of ACE2-expressing cell 3,4. Furthermore, ACE2 was proposed to be an ISG, on the basis of its inducible expression in cells treated with interferons (IFNs) or infected by viruses that induce IFN responses, such as influenza 4,5. These findings implied that the induction of ACE2 expression in IFN-high conditions could result in an amplified risk of SARS-CoV-2 infection 4,5. Concerns could also be raised about possible ACE2-inducing side effects of IFN-based treatments proposed for COVID-19 (refs. 6-9). ACE2 plays multiple roles in normal physiological conditions and as part of the host tissue-protective machinery in damaging conditions, including viral infections. As a terminal carboxypeptidase, ACE2 cleaves a single carboxy-terminal residue from peptide hormones such as angiotensin II and des-Arg9-bradykinin. ACE and ACE2 belong to the renin-angiotensin-aldosterone system, which regulates blood pressure and fluid-electrolyte balance; dysfunction of this system contributes to comorbidities in COVID-19 (refs. 10,11). des-Arg9-bradykinin is generated from bradykinin and belongs to the kallikrein-kinin system, which is critical in regulating vascular leakage and pulmonary edema, early signs of severe COVID-19 (refs. 12,13). High plasma angiotensin II levels were found to be responsible for coronavirus-associated acute respiratory distress syndrome (ARDS), lung damage and high mortality in mouse models 14,15 and as a predictor of lethality in avian influenza in humans 16,17. In the same conditions, ACE2, which decreases the levels of angiotensin II, was identified as a protective factor. The hijacking of the normal host tissue-protective machinery guarded by ACE2 was suggested as a mechanism through which SARS-CoV-2 could infect more cells 4,5. Thus, it is critically important to identify factors affecting ACE2 expression in normal physiological processes and during viral infections and associated pathologies, such as in COVID-19. Herein, aiming to explore the IFN-inducible expression of ACE2 and its role in SARS-CoV-2 infection, we identified a novel, truncated isoform of ACE2, which we designate as dACE2. We then showed that dACE2, but not ACE2, is induced in various human cell types by IFNs and viruses; this information is important to consider for future therapeutic strategies and understanding COVID-19 susceptibility and outcomes. Results dACE2 is a novel inducible isoform of ACE2. To address the extent to which IFNs induce the expression of ACE2 in human cells, we used our existing RNA-seq dataset (NCBI Sequence Read Archive (SRA): PRJNA512015) of a breast cancer cell line T47D infected with Sendai virus (SeV), known to be a strong inducer of IFNs and ISGs 18-20. IFNs were not expressed in T47D cells at baseline, but SeV strongly induced expression of IFNB1, a type I IFN, an...

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Background To date, only monoclonal antibodies have been shown to be effective for outpatients with COVID-19. Interferon lambda-1 is a type III interferon involved in innate antiviral responses with activity against respiratory pathogens. We aimed to investigate the safety and efficacy of peginterferon lambda in the treatment of outpatients with mild-to-moderate COVID-19. MethodsIn this double-blind, placebo-controlled trial, outpatients with laboratory-confirmed COVID-19 were randomly assigned to a single subcutaneous injection of peginterferon lambda 180 µg or placebo within 7 days of symptom onset or first positive swab if asymptomatic. Participants were randomly assigned (1:1) using a computergenerated randomisation list created with a randomisation schedule in blocks of four. At the time of administration, study nurses received a sealed opaque envelope with the treatment allocation number. The primary endpoint was the proportion of patients who were negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on day 7 after the injection, analysed by a χ² test following an intention-to-treat principle. Prespecified analysis of the primary endpoint, adjusted for baseline viral load, using bivariate logistic regression was done. The trial is now complete. This trial is registered with ClinicalTrials.gov, NCT04354259.Findings Between May 18, and Sept 4, 2020, we recruited 30 patients per group. The decline in SARS-CoV-2 RNA was greater in those treated with peginterferon lambda than placebo from day 3 onwards, with a difference of 2•42 log copies per mL at day 7 (p=0•0041). By day 7, 24 (80%) participants in the peginterferon lambda group had an undetectable viral load, compared with 19 (63%) in the placebo group (p=0•15). After controlling for baseline viral load, patients in the peginterferon lambda group were more likely to have undetectable virus by day 7 than were those in the placebo group (odds ratio [OR] 4•12 [95% CI 1•15-16•73; p=0•029). Of those with baseline viral load above 10⁶ copies per mL, 15 (79%) of 19 patients in the peginterferon lambda group had undetectable virus on day 7, compared with six (38%) of 16 in the placebo group (OR 6•25 [95% CI 1•49-31•06]; p=0•012). Peginterferon lambda was well tolerated, and adverse events were similar between groups with mild and transient aminotransferase, concentration increases more frequently observed in the peginterferon lambda group. Two individuals met the threshold of grade 3 increase, one in each group, and no other grade 3 or 4 laboratory adverse events were reported.Interpretation Peginterferon lambda accelerated viral decline in outpatients with COVID-19, increasing the proportion of patients with viral clearance by day 7, particularly in those with high baseline viral load. Peginterferon lambda has potential to prevent clinical deterioration and shorten duration of viral shedding.

We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30–39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.