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The efficacy and safety of coronavirus disease 2019 (COVID-19) booster vaccines remain limited. We investigated the immunogenicity and adverse events of the third dose of mRNA vaccines in healthy adults.Volunteers vaccinated with two doses of the adenoviral vaccine (ChAdOx1) 12 weeks before were administered with an mRNA COVID-19 vaccine. These were divided into three groups, full-dose mRNA-1273 (group 1); half-dose mRNA-1273 (group 2); and full-dose BNT-162b2 (group 3). Primary outcomes included serum anti-SARS-CoV-2 spike immunoglobulin G (IgG) titers and neutralizing antibody titers against B.1.1.7 (alpha), B.1.617.2 (delta), and B.1.1.529 (omicron) variants. Secondary outcomes included the evaluation of humoral and cellular immunity and vaccine-associated adverse events after the boost.Totally 300 participants were recruited, and 298 participants were enrolled. For all three groups, an increase in anti-SARS-CoV-2 spike IgG geometric mean titers (30.12- to 71.80-fold) and neutralizing antibody titers against the alpha variant (69.80- to 173.23-folds), delta variant (132.69- to 324.63-folds), and omicron variant (135.36- to 222.37-folds) were observed on day 28. All groups showed robust T- and B-cell responses after boosting. Adverse events were overall mild and transient but with higher prevalence and severity in group 1 participants than in other groups.Third dose mRNA COVID-19 vaccines markedly enhanced cellular and humoral responses and were safe. Immunological responses and adverse events were higher in individuals receiving the full-dose mRNA-1273 vaccine, followed by a half-dose mRNA-1273 vaccine and BNT-162b2 vaccine.

Type I interferons are important antiviral cytokines, but prolonged interferon production is detrimental to the host. The TLR3-driven immune response is crucial for mammalian antiviral immunity, and its intracellular localization determines induction of type I interferons; however, the mechanism terminating TLR3 signaling remains obscure. Here, we show that the E3 ubiquitin ligase ZNRF1 controls TLR3 sorting into multivesicular bodies/lysosomes to terminate signaling and type I interferon production. Mechanistically, c-Src kinase activated by TLR3 engagement phosphorylates ZNRF1 at tyrosine 103, which mediates K63-linked ubiquitination of TLR3 at lysine 813 and promotes TLR3 lysosomal trafficking and degradation. ZNRF1-deficient mice and cells are resistant to infection by encephalomyocarditis virus and SARS-CoV-2 because of enhanced type I interferon production. However, Znrf1−/− mice have exacerbated lung barrier damage triggered by antiviral immunity, leading to enhanced susceptibility to respiratory bacterial superinfections. Our study highlights the c-Src–ZNRF1 axis as a negative feedback mechanism controlling TLR3 trafficking and the termination of TLR3 signaling.

Analyses of the genomic diversity of SARS-CoV-2 found that some sites across the genome appear to have mutated independently multiple times with frequency significantly higher than four-fold sites, which can be either due to mutational bias, i.e., elevated mutation rate in some sites of the genome, or selection of the variants due to antagonistic pleiotropy, a condition where mutations increase some components of fitness at a cost to others. To examine how different forces shaped evolution of SARS-CoV-2 in 2020–2021, we analyzed a large set of genome sequences (~ 2 million). Here we show that while evolution of SARS-CoV-2 during the pandemic was largely mutation-driven, a group of nonsynonymous changes is probably maintained by antagonistic pleiotropy. To test this hypothesis, we studied the function of one such mutation, spike M1237I. Spike I1237 increases viral assembly and secretion, but decreases efficiency of transmissionin vitro. Therefore, while the frequency of spike M1237I may increase within hosts, viruses carrying this mutation would be outcompeted at the population level. We also discuss how the antagonistic pleiotropy might facilitate positive epistasis to promote virus adaptation and reconcile discordant estimates of SARS-CoV-2 transmission bottleneck sizes in previous studies.

We presented the clinical course and immune responses of a well-controlled HIV-positive patient with COVID-19. The clinical presentation and antibody production to SARS-CoV-2 were similar to other COVID-19 patients without HIV infection. Neutralizing antibody reached a plateau from 26th to 47th day onset but decreased on 157th day after symptoms.

Patients recovering from COVID-19 may need vaccination against SARS-CoV-2 because acquired immunity from primary infection may wane, given the emergence of new SARS-CoV-2 variants. Understanding the trends of anti-spike IgG and neutralizing antibody titers in patients recovering from COVID-19 may inform the decision made on the appropriate interval between recovery and vaccination.Participants aged 20 years or older and diagnosed with COVID-19 between January and December, 2020 were enrolled. Serum specimens were collected every three months from 10 days to 12 months after the onset of symptom for determinations of anti-spike IgG and neutralizing antibody titers against SARS-CoV-2 Wuhan strain with D614G mutation, alpha, gamma and delta variants.Of 19 participants, we found a decreasing trend of geometric mean titers of anti-spike IgG from 560.9 to 217 and 92 BAU/mL after a 4-month and a 7-month follow-up, respectively. The anti-spike IgG titers declined more quickly in the ten participants with severe or critical disease than the nine participants with only mild to moderate disease between one month and seven months after SARS-CoV-2 infection (-8.49 vs - 2.34-fold, p 0.001). The neutralizing activity of the convalescent serum specimens collected from participants recovering from wild-type SARS-CoV-2 infection against different variants was lower, especially against the delta variants (p 0.01 for each variant with Wuhan strain as reference).Acquired immunity from primary infection with SARS-CoV-2 waned within 4-7 months in COVID-19 patients, and neutralizing cross-activities against different SARS-CoV-2 variants were lower compared with those against wild-type strain.

An increasing body of evidence emphasizes the role of metabolic reprogramming in immune cells to fight off infections. However, little is known about the regulation of metabolite transporters that facilitate and support metabolic demands. In this study, we found that equilibrative nucleoside transporter 3 (ENT3) expression is part of the innate immune response, and is rapidly upregulated upon bacterial and viral infection. The transcription of ENT3 is directly under the regulation of IFN-induced signaling, positioning this metabolite transporter as an Interferon-stimulated gene (ISG). Moreover, we unveil that several viruses, including SARS-CoV2, require ENT3 to facilitate their entry into the cytoplasm. The removal or suppression of ENT3 expression is sufficient to significantly decrease viral replication in vitro and in vivo.

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc–decorated state (S MG ) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from S MG -immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Neutralizing antibodies (NAbs) are believed to be promising prophylactic and therapeutic treatment against the coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we reported two mouse monoclonal antibodies 7 Eb-4G and 1Ba-3H that specifically recognized the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein without exhibiting cross-reactivity with the S proteins of SARS-CoV and MERS-CoV. The binding epitopes of 7 Eb-4G and 1Ba-3H were respectively located in the regions of residues 457-476 and 477-496 in the S protein. Only 1Ba-3H exhibited the neutralizing activity for preventing the pseudotyped lentivirus from binding to the angiotensin-converting enzyme 2 (ACE2)-transfected HEK293T cells. The competitive ELISA further showed that 1Ba-3H interfered with the binding between RBD and ACE2. Epitope mapping experiments demonstrated that a single alanine replacement at residues 480, 482, 484, 485, and 488-491 in the RBD abrogated 1Ba-3H binding. 1Ba-3H exhibited the neutralizing activity against the wild-type, Alpha, Delta, and Epsilon variants of SARS-CoV-2, but lost the neutralizing activity against Gamma variant in the plaque reduction assay. On the contrary, 1Ba-3H enhanced the cellular infection of Gamma variant in a dose-dependent manner. Our findings suggest that the antibody-dependent enhancement of infection mediated by the RBD-specific antibody for different SARS-CoV-2 variants must be considered while developing the NAb.

Remdesivir, a prodrug targeting RNA-dependent-RNA-polymerase, and cyclosporine, a calcineurin inhibitor, individually exerted inhibitory activity against human coronavirus OC43 (HCoV-OC43) in HCT-8 and MRC-5 cells at EC50 values of 96 ± 34 ∼ 85 ± 23 nM and 2,920 ± 364 ∼ 4,419 ± 490 nM, respectively. When combined, these two drugs synergistically inhibited HCoV-OC43 in both HCT-8 and MRC-5 cells assayed by immunofluorescence assay (IFA). Remdesivir and cyclosporine also separately reduced IL-6 production induced by HCoV-OC43 in human lung fibroblasts MRC-5 cells with EC50 values of 224 ± 53 nM and 1,292 ± 352 nM, respectively; and synergistically reduced it when combined. Similar trends were observed for SARS-CoV-2, which were 1) separately inhibited by remdesivir and cyclosporine with respective EC50 values of 3,962 ± 303 nM and 7,213 ± 143 nM by IFA, and 291 ± 91 nM and 6,767 ± 1,827 nM by a plaque-formation assay; and 2) synergistically inhibited by their combination, again by IFA and plaque-formation assay. Collectively, these results suggest that the combination of remdesivir and cyclosporine merits further study as a possible treatment for COVID-19 complexed with a cytokine storm.

Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 (S-G614 and S-D614, respectively). The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. IMPORTANCE Analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time has identified the D614G substitution in spike (S) as the most prevalent expanding variant worldwide, which might confer a selective advantage in transmission. Several studies showed that the D614G variant replicates and transmits more efficiently than the wild-type virus, but the mechanism is unclear. By comparing 18 virus isolates containing S with either D614 or G614, we found significantly higher virus titers in association with higher furin protease-mediated cleavage of S, an event that promotes syncytium formation and virus infectivity, in the S-G614 viruses. The effect of the D614G substitution on furin-mediated S cleavage and the resulting enhancement of the syncytium phenotype has been validated in S-expressing cells. This study suggests a possible effect of the D614G substitution on S of SARS-CoV-2; the antiviral effect through targeting furin protease is worthy of being investigated in proper animal models.

A major challenge to end the pandemic caused by SARS-CoV-2 is to develop a broadly protective vaccine. As the key immunogen, the spike protein is frequently mutated with conserved epitopes shielded by glycans. Here, we reveal that spike glycosylation has site-differential effects on viral infectivity and lung epithelial cells generate spike with more infective glycoforms. Compared to the fully glycosylated spike, immunization of spike protein with N-glycans trimmed to the monoglycosylated state (Smg) elicits stronger immune responses and better protection for hACE2 transgenic mice against variants of concern. In addition, a broadly neutralizing monoclonal antibody was identified from the Smg immunized mice, demonstrating that removal of glycan shields to better expose the conserved sequences is an effective and simple approach to broad-spectrum vaccine development.One-Sentence SummaryRemoving glycan shields to expose conserved epitopes is an effective approach to develop a broad-spectrum SARS-CoV-2 vaccine.

Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CLpro) and papain-like protease (PLpro) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease., Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CLpro) and papain-like protease (PLpro) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease. In this study, 3CLpro and PLpro assay platforms were established, and their substrate specificities were characterized. The assays were used to screen collections of 1,068 and 2,701 FDA-approved drugs. After excluding the externally used drugs which are too toxic, we totally identified 12 drugs as 3CLpro inhibitors and 36 drugs as PLpro inhibitors active at 10 μM. Among these inhibitors, six drugs were found to suppress SARS-CoV-2 with the half-maximal effective concentration (EC50) below or close to 10 μM. This study enhances our understanding on the proteases and provides FDA-approved drugs for prevention and/or treatment of COVID-19.

Niclosamide, a widely-used anthelmintic drug, inhibits SARS-CoV-2 virus entry through TMEM16F inhibition and replication through autophagy induction, but the relatively high cytotoxicity and poor oral bioavailability limited its application. We synthesized 22 niclosamide analogues of which compound 5 was found to exhibit the best anti-SARS-CoV-2 efficacy (IC

Entry inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to control the outbreak of coronavirus disease 2019 (COVID-19). This study developed a robust and straightforward assay that detected the molecular interaction between the receptor-binding domain (RBD) of viral spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor in just 10 min. A drug library of 1068 approved compounds was used to screen for SARS-CoV2 entry inhibition, and 9 active drugs were identified as specific pseudovirus entry inhibitors. A plaque reduction neutralization test using authentic SARS-CoV-2 virus in Vero E6 cells confirmed that 2 of these drugs (Etravirine and Dolutegravir) significantly inhibited the infection of SARS-CoV-2. With molecular docking, we showed that both Etravirine and Dolutegravir are preferentially bound to primary ACE2-interacting residues on the RBD domain, implying that these two drug blocks may prohibit the viral attachment of SARS-CoV-2. We compared the neutralizing activities of these entry inhibitors against different pseudoviruses carrying spike proteins from alpha, beta, gamma, and delta variants. Both Etravirine and Dolutegravir showed similar neutralizing activities against different variants, with EC50 values between 4.5 to 5.8 nM for Etravirine and 10.2 to 22.9 nM for Dolutegravir. These data implied that Etravirine and Dolutegravir may serve as general spike inhibitors against dominant viral variants of SARS-CoV-2.

Tylophorine-based compounds and natural cardiotonic steroids (cardenolides and bufadienolides) are two classes of transmissible gastroenteritis coronavirus inhibitors, targeting viral RNA and host cell factors, respectively We tested both types of compounds against two types of coronaviruses, to compare and contrast their antiviral properties, and with view to their further therapeutic development Examples of both types of compounds potently inhibited the replication of both feline infectious peritonitis virus and human coronavirus OC43 with EC50 values of up to 8 and 16 nM, respectively Strikingly, the tylophorine-based compounds tested inhibited viral yields of HCoV-OC43 to a much greater extent (7–8 log magnitudes of p f u /ml) than the cardiotonic steroids (about 2–3 log magnitudes of p f u /ml), as determined by end point assays Based on these results, three tylophorine-based compounds were further examined for their anti-viral activities on two other human coronaviruses, HCoV-229E and SARS-CoV-2 These three tylophorine-based compounds inhibited HCoV-229E with EC50 values of up to 6 5 nM, inhibited viral yields of HCoV-229E by 6–7 log magnitudes of p f u /ml, and were also found to inhibit SARS-CoV-2 with EC50 values of up to 2 5–14 nM In conclusion, tylophorine-based compounds are potent, broad-spectrum inhibitors of coronaviruses including SARS-CoV-2, and could be used for the treatment of COVID-19 © Copyright © 2020 Yang, Lee, Hsu, Jan, Lin, Chang, Peng, Yang, Liang, Liao, Chao, Pang, Kao, Huang, Lin, Chang, Niu, Wu, Sytwu, Chen and Lee

Development of specific antivirals is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infections. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validated cleavage at a putative furin substrate motif at SARS-CoV-2 spike by expressing it in VeroE6 cells and found prominent syncytium formation. Both cleavage and syncytium were abolished by treatment with furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein but not by transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein showed antiviral effects in SARS-CoV-2-infected cells by decreasing viral production and cytopathic effects. Further analysis revealed that, similar to camostat, CMK blocks virus entry, but it further suppresses the cleavage of spike and syncytium. Naphthofluorescein instead acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may become promising antivirals for prevention and treatment of SARS-CoV-2 infections., Graphical Abstract, Highlights ● The furin cleavage site in the SARS-CoV-2 spike protein mediates syncytium formation. ● The SARS-CoV-2 spike-mediated syncytium is suppressed by specific furin inhibitors. ● Furin inhibitors block SARS-CoV-2 viral entry and viral replication. ● Furin inhibitors are potential antivirals for SARS-CoV-2 infection and pathogenesis., Development of effective antivirals is an urgent unmet need for SARS-CoV-2 infections. Cheng et al. find that the cleavage of the furin substrate site in the viral spike protein is critical for viral production and cytopathic effects. Two inhibitors targeting furin are potential antivirals to control SARS-CoV-2 infection and pathogenesis.

A metal nanoparticle composite, namely TPNT1, which contains Au-NP (1 ppm), Ag-NP (5 ppm), ZnO-NP (60 ppm) and ClO2 (42.5 ppm) in aqueous solution was prepared and characterized by spectroscopy, transmission electron microscopy, dynamic light scattering analysis and potentiometric titration. Based on the in vitro cell-based assay, TPNT1 inhibited six major clades of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with effective concentration within the range to be used as food additives. TPNT1 was shown to block viral entry by inhibiting the binding of SARS-CoV-2 spike proteins to the angiotensin-converting enzyme 2 (ACE2) receptor and to interfere with the syncytium formation. In addition, TPNT1 also effectively reduced the cytopathic effects induced by human (H1N1) and avian (H5N1) influenza viruses, including the wild-type and oseltamivir-resistant virus isolates. Together with previously demonstrated efficacy as antimicrobials, TPNT1 can block viral entry and inhibit or prevent viral infection to provide prophylactic effects against both SARS-CoV-2 and opportunistic infections.

Cell culture remains as the golden standard for primary isolation of viruses in clinical specimens. In the current practice, researchers have to recognize the cytopathic effects (CPE) induced by virus infection and subsequently use virus-specific monoclonal antibody to confirm the presence of virus. Considering the broad applications of neural network in various fields, we aimed to utilize convolutional neural networks (CNN) to shorten the timing required for CPE identification and to improve the assay sensitivity. Based on the characteristics of influenza-induced CPE, a CNN model with larger sizes of filters and max-pooling kernels was constructed in the absence of transfer learning. A total of 601 images from mock-infected and influenza-infected MDCK cells were used to train the model. The performance of the model was tested by using extra 400 images and the percentage of correct recognition was 99.75%. To further examine the limit of our model in evaluating the changes of CPE overtime, additional 1190 images from a new experiment were used and the recognition rates at 16 hour (hr), 28 hr, and 40 hr post virus infection were 71.80%, 98.25%, and 87.46%, respectively. The specificity of our model, examined by images of MDCK cells infected by six other non-influenza viruses, was 100%. Hence, a simple CNN model was established to enhance the identification of influenza virus in clinical practice., Author summary Observation of cytopathic effects (CPE) induced by virus infection is a practical method to determine the prsence of viruses in the clinical specimens. However, CPE observation is labor-intensive and time-consuming because it requires medical examiner to inspect cell morphology changes for a period of time. Here, Convolutional Neural Networks (CNN) was applied to improve the disadvantage of CPE observation by using influenza virus as an example. To reduce the requirement for large image input of every clinical test, small amount of data was used to train our CNNs model without transfer learning and the trained model was examined with testing image data taken at 25hr post virus infection. The recognition of testing data shows that the model can identify CPE at 25hr and the high specificity of the model can differentiate the CPE induced by influenza viruses from those by other non-influenza viruses. The limit of our model was further examined by more experimental data of influenza-induced and mock-infected images, and the result shows our model can detect the slight changes at the initial stage of CPE development. Hence, our deep CNN model can significantly shorten the timing required to identify virus-induced cytopathic effects.

The recent emerging coronavirus, SARS-CoV-2, has been rapidly and widely spread and causing an ongoing viral pneumonia outbreak worldwide. It has been observed that SARS-CoV-2 patients show a rather long and asymptomatic incubation time. We characterized the abilities to induce and to response to IFNβ/IFNλ1 of two or our clinical isolates, SARS-CoV-2/NTU01/TWN/human/2020 and SARS-CoV-2/NTU02/TWN/human/2020, which exhibit only two amino acid differences over the ∼30kb viral genome. We found that both isolates may infect Huh7, A549 and Calu-3 cells, yet the RIG-I-like receptor-dependent antiviral signaling was poorly induced in these cells in the early infections. Unexpectedly, we found that the intracellular vRNA levels of these isolates were sustained upon to type I/III IFN treatments, and this phenotype was more pronounced in the Taiwan/NTU01/2020 isolate. The type I/III IFN responses are antiviral but partially proviral in the case of SARS-CoV-2 infections. Poor induction and response to innate immunity may contribute to destitute neutralization index of the antibody produced, and indeed we found that the patient serum could not efficiently neutralize SARS-CoV-2 virions. With better understandings of the interplay between SARS-CoV-2 and the host antiviral innate immunity, our report may provide new insights for the regimen of therapies for SARS-CoV-2 infected patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent for the outbreak of coronavirus disease 2019 (COVID-19). This global pandemic is now calling for efforts to develop more effective COVID-19 therapies. Here we use a host-directed approach, which focuses on cellular responses to diverse small-molecule treatments, to identify potentially effective drugs for COVID-19. This framework looks at the ability of compounds to elicit a similar transcriptional response to IFN-β, a type I interferon that fails to be induced at notable levels in response to SARS-CoV-2 infection. By correlating the perturbation profiles of ~3,000 small molecules with a high-quality signature of IFN-β-responsive genes in primary normal human bronchial epithelial cells, our analysis revealed four candidate COVID-19 compounds, namely homoharringtonine, narciclasine, anisomycin, and emetine. We experimentally confirmed that the predicted compounds significantly inhibited SARS-CoV-2 replication in Vero E6 cells at nanomolar, relatively non-toxic concentrations, with half-maximal inhibitory concentrations of 165.7 nM, 16.5 nM, and 31.4 nM for homoharringtonine, narciclasine, and anisomycin, respectively. Together, our results corroborate a host-centric strategy to inform protective antiviral therapies for COVID-19., Systems biology; COVID-19; Host-directed therapy; Type I interferon; Drug repurposing; Bioinformatics; Infectious disease; Transcriptomics; Pharmaceutical science

We describe the clinical course and virological characteristics of the first H7N9 influenza case in a Taiwanese patient; this patient had detectable viruses in the airway for 2 weeks, during which time an oseltamivir resistance-associated R292K mutation rapidly emerged. Anti-H7N9 antibody was detected 21 days after onset of symptoms, when H7N9 viral load declined significantly.