Search

Background and objective Few head-to-head evaluations of immune responses to different vaccines have been reported. Methods Surrogate virus neutralization test (sVNT) antibody levels of adults receiving either two doses of BNT162b2 (n = 366) or CoronaVac (n = 360) vaccines in Hong Kong were determined. An age-matched subgroup (BNT162b2 [n = 49] vs. CoronaVac [n = 49]) was tested for plaque reduction neutralization (PRNT) and spike-binding antibody and T-cell reactivity in peripheral blood mononuclear cells. Results One month after the second dose of vaccine, BNT162b2 elicited significantly higher PRNT50 , PRNT90 , sVNT, spike receptor binding, spike N-terminal domain binding, spike S2 domain binding, spike FcR binding and antibody avidity levels than CoronaVac. The geometric mean PRNT50 titres in those vaccinated with BNT162b2 and CoronaVac vaccines were 251.6 and 69.45, while PRNT90 titres were 98.91 and 16.57, respectively. All of those vaccinated with BNT162b2 and 45 (91.8%) of 49 vaccinated with CoronaVac achieved the 50% protection threshold for PRNT90. Allowing for an expected seven-fold waning of antibody titres over 6 months for those receiving CoronaVac, only 16.3% would meet the 50% protection threshold versus 79.6% of BNT162b2 vaccinees. Age was negatively correlated with PRNT90 antibody titres. Both vaccines induced SARS-CoV-2-specific CD4+ and CD8+ T-cell responses at 1 month post-vaccination but CoronaVac elicited significantly higher structural protein-specific CD4+ and CD8+ T-cell responses. Conclusion Vaccination with BNT162b2 induces stronger humoral responses than CoronaVac. CoronaVac induces higher CD4+ and CD8+ T-cell responses to the structural protein than BNT162b2.

The increasing numbers of infected cases of coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) poses serious threats to public health and the global economy. Most SARS‐CoV‐2 neutralizing antibodies target the receptor binding domain (RBD) and some the N‐terminal domain (NTD) of the spike protein, which is the major antigen of SARS‐CoV‐2. While the antibody response to RBD has been extensively characterized, the antigenicity and immunogenicity of the NTD protein are less well studied. Using 227 plasma samples from COVID‐19 patients, we showed that SARS‐CoV‐2 NTD‐specific antibodies could be induced during infection. As compared to the results of SARS‐CoV‐2 RBD, the serological response of SARS‐CoV‐2 NTD is less cross‐reactive with SARS‐CoV, a pandemic strain that was identified in 2003. Furthermore, neutralizing antibodies are rarely elicited in a mice model when NTD is used as an immunogen. We subsequently demonstrate that NTD has an altered antigenicity when expressed alone. Overall, our results suggest that while NTD offers a supplementary strategy for serology testing, it may not be suitable as an immunogen for vaccine development., Receptor binding domain (RBD) of the SARS‐CoV‐2 spike protein has been extensively studied in functional antibody screening and COVID‐19 vaccine design. To explore the function of N‐terminal domain (NTD) of spike protein, we reveal that NTD is a supplementary antigen for serology testing, but not a suitable immunogen for vaccine design.

Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) (ISG15) is a ubiquitin-like modifier induced during infections and involved in host defense mechanisms. Not surprisingly, many viruses encode deISGylating activities to antagonize its effect. Here we show that infection by Zika, SARS-CoV-2 and influenza viruses induce ISG15-modifying enzymes. While influenza and Zika viruses induce ISGylation, SARS-CoV-2 triggers deISGylation instead to generate free ISG15. The ratio of free versus conjugated ISG15 driven by the papain-like protease (PLpro) enzyme of SARS-CoV-2 correlates with macrophage polarization toward a pro-inflammatory phenotype and attenuated antigen presentation. In vitro characterization of purified wild-type and mutant PLpro revealed its strong deISGylating over deubiquitylating activity. Quantitative proteomic analyses of PLpro substrates and secretome from SARS-CoV-2-infected macrophages revealed several glycolytic enzymes previously implicated in the expression of inflammatory genes and pro-inflammatory cytokines, respectively. Collectively, our results indicate that altered free versus conjugated ISG15 dysregulates macrophage responses and probably contributes to the cytokine storms triggered by SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR–confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20–40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti–SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients’ plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.

Summary: Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses. : Jahan et al. describe the role of Otub1, a deubiquitylase that is induced during virus infection in an interferon-dependent manner to regulate optimal signaling via the RIG-I pathway. Influenza A virus NS1 is able to bind and degrade newly synthesized Otub1 as a means to evade the innate immune response. Keywords: deubiquitylases, RIG-I signaling, ubiquitylation, innate immune response, RNA virus, influenza A, viral subversion strategies

Human coronavirus NL63 (HCoV-NL63) is primarily associated with common cold in children, elderly and immunocompromised individuals. Outbreaks caused by HCoV-NL63 are rare. Here we report a cluster of HCoV-NL63 cases with severe lower respiratory tract infection that arose in Guangzhou, China, in 2018. Twenty-three hospitalized children were confirmed to be HCoV-NL63 positive, and most of whom were hospitalized with severe pneumonia or acute bronchitis. Whole genomes of HCoV-NL63 were obtained using next-generation sequencing. Phylogenetic and single amino acid polymorphism analyses showed that this outbreak was associated with two subgenotypes (C3 and B) of HCoV-NL63. Half of patients were identified to be related to a new subgenotype C3. One unique amino acid mutation at I507 L in spike protein receptor binding domain (RBD) was detected, which segregated this subgenotype C3 from other known subgenotypes. Pseudotyped virus bearing the I507 L mutation in RBD showed enhanced entry into host cells as compared to the prototype virus. This study proved that HCoV-NL63 was undergoing continuous mutation and has the potential to cause severe lower respiratory disease in humans.

BackgroundPoor immunogenicity and antibody waning were found in vaccinees of CoronaVac. There is lack of randomized controlled trial (RCT) data to compare the immunogenicity and safety of schedules using homologous and heterologous vaccine as a booster dose.MethodsWe randomly assigned adults who had received 2 doses of CoronaVac with low antibody response to receive an additional booster dose of either BNT162b2 or CoronaVac. The local and systemic adverse reactions were recorded. Levels of SARS-CoV-2 neutralizing and spike binding antibody in plasma were measured.FindingsAt one month after the third dose of vaccine, BNT162b2 vaccines elicited significantly higher surrogate virus neutralizing test (sVNT), spike receptor binding, spike N terminal domain binding, spike S2 domain binding levels than CoronaVac. More participants from the BNT162b2 group reported injection site pain and swelling as well as fatigue and muscle pain than those who received CoronaVac as the third dose. The mean results of the sVNT against the wild type, beta, gamma and delta variants in the BNT162b2 boosted group was 96.83%, 92.29%, 92.51% and 95.33% respectively which were significantly higher than the CoronaVac boosted group (Wild type: 57.75%; Beta: 38.79 %; Gamma: 32.22%; Delta: 48.87%)ConclusionOur RCT study shows that BNT162b2 booster dose for those people who poorly responded to the previous vaccination of CoronaVac is significantly more immunogenic than a CoronaVac booster. BNT162b2 also elicits higher levels of SARS-CoV-2 specific neutralizing antibodies to different variants of concern. The adverse reactions were only mild and short-lived.At a Glance CommentaryScientific Knowledge on the SubjectPoor immunogenicity and antibody waning were found in vaccinees of CoronaVac. There is lack of randomized controlled trial (RCT) data to compare the immunogenicity and safety of schedules using homologous and heterologous vaccine as a booster dose.What This Study Adds to the FieldOur RCT study shows that BNT162b2 booster dose for those people who poorly responded to the previous vaccination of CoronaVac is significantly more immunogenic than a CoronaVac booster. The adverse reactions were only mild and short-lived.

BackgroundFew head-to-head evaluations of immune responses to difference vaccines have been reported.MethodsSurrogate virus neutralization test (sVNT) antibody levels of adults receiving either 2 doses of BNT162b2 (n=366) or CoronaVac (n=360) vaccines in Hong Kong were determined. An age-matched subgroup (BNT162b2 (n=49) vs CoronaVac (n=49)) were tested for plaque reduction neutralizing (PRNT) and spike binding antibody and T cell reactivity in peripheral blood mononuclear cells (PBMC).FindingsOne month after the second dose of vaccine, BNT162b2 elicited significantly higher PRNT50, PRNT90, sVNT, spike receptor binding, spike N terminal domain binding, spike S2 domain binding, spike FcR binding and antibody avidity levels than CoronaVac. The geometric mean PRNT50 titres in those vaccinated with BNT162b2 and CoronaVac vaccines were 251.6 and 69.45 while PRNT90 titres were 98.91 and 16.57, respectively. All of those vaccinated with BNT162b2 and 45 (91.8%) of 49 vaccinated with CoronaVac achieved the 50% protection threshold for PRNT90. Allowing for an expected seven-fold waning of antibody titres over six months for those receiving CoronaVac, only 16.3% would meet the 50% protection threshold versus 79.6% of BNT162b2 vaccinees. Age was negatively correlated with PRNT90 antibody titres. Both vaccines induced SARS-CoV-2 specific CD4+ and CD8+ T cell responses at 1-month post-vaccination but CoronaVac elicited significantly higher structural protein-specific CD4+ and CD8+ T cell responses.ConclusionVaccination with BNT162b2 induces stronger humoral responses than CoronaVac. CoronaVac induce higher CD4+ and CD8+ T cell responses to the structural protein than BNT162b2.Summary At a GlanceThrough the head-to-head comparison, vaccination with BNT162b2 induces significantly higher levels of SARS-CoV-2 specific binding and neutralizing antibody responses when compared to CoronaVac. CoronaVac induce higher CD4+ and CD8+ T cell responses to the structural protein than BNT162b2.

Significance The absence of zoonotic MERS-CoV in Africa in spite of an abundance of MERS-CoV–infected dromedaries has remained an enigma. We demonstrate that geographically and genetically distinct viruses from Africa have low replication competence in the human lung, providing a possible explanation for the absence of severe MERS disease in Africa. The findings suggest that MERS-CoV now entrenched in the Arabian Peninsula has acquired increased pathogenic potential for humans. We demonstrate that the spike protein contributes to this phenotypic difference. If pathogenic clade B viruses from the Arabian Peninsula are introduced into Africa, they are likely to become dominant, as they have in the Arabian Peninsula, and to be associated with adverse health impacts in Africa and increased pandemic threat., Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV–infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Background Highly pathogenic avian influenza (HPAI)-H7N9 virus arising from low pathogenic avian influenza (LPAI)-H7N9 virus with polybasic amino acid substitutions in the hemagglutinin was detected in 2017. Methods We compared the tropism, replication competence, and cytokine induction of HPAI-H7N9, LPAI-H7N9, and HPAI-H5N1 in ex vivo human respiratory tract explants, in vitro culture of human alveolar epithelial cells (AECs) and pulmonary microvascular endothelial cells (HMVEC-L). Results Replication competence of HPAI- and LPAI-H7N9 were comparable in ex vivo cultures of bronchus and lung. HPAI-H7N9 predominantly infected AECs, whereas limited infection was observed in bronchus. The reduced tropism of HPAI-H7N9 in bronchial epithelium may explain the lack of human-to-human transmission despite a number of mammalian adaptation markers. Apical and basolateral release of virus was observed only in HPAI-H7N9- and H5N1-infected AECs regardless of infection route. HPAI-H7N9, but not LPAI-H7N9 efficiently replicated in HMVEC-L. Conclusions Our findings demonstrate that a HPAI-H7N9 virus efficiently replicating in ex vivo cultures of human bronchus and lung. The HPAI-H7N9 was more efficient at replicating in human AECs and HMVEC-L than LPAI-H7N9 implying that endothelial tropism may involve in pathogenesis of HPAI-H7N9 disease.

Mutations in the polymerase basic 2 (PB2) gene of avian influenza viruses are important signatures for their adaptation to mammalian hosts. Various adaptive mutations have been identified around the 627 and nuclear localization sequence (NLS) domains of PB2 protein, and these mutations contribute to the replicative ability of avian influenza viruses. However, few studies have focused on adaptive mutations in other regions of PB2. In this study, we investigated the functional roles of the D253N mutation in PB2 in an H9N2 virus. This mutation was found to affect an amino acid residue in the middle domain of the PB2 protein. The virus with the D253N mutation showed higher polymerase activity and transiently increased viral replication in human cells. However, the mutant did not show significant differences in viral replication in the respiratory tract of mice upon infection. Our results supported that the D253N mutation in the middle domain of PB2, similar to mutations at the 627 and NLS domains, specifically contributed to the replication of avian influenza viruses in human cells.

Outbreaks of avian influenza virus continue to pose threats to human health. Animal models such as the mouse, ferret, and macaque are used to understand the pathogenesis of avian influenza virus infection in humans. We previously reported that the tree shrew (Tupaia belangeri, family Tupaiidae), which is regarded as a “low-level primate”, has α2,3- and α2,6-linked sialic acid receptor distributions similar to those of humans and is potentially a useful mammalian model for studying mild human influenza (H1N1) virus infection. In this study, we used the tree shrew experimental model to investigate the pathogenesis of avian influenza A (H9N2) virus infection and the effect of the E627K mutation in the PB2 gene, an adaptation to mammalian hosts. Evidence of disease, virus titers in the upper and lower respiratory tract, histopathology and induction of proinflammatory cytokines are described. We also established ex vivo culture models of tree shrew respiratory tissues to study the tropism and replication of the H9N2 virus. Our results demonstrated that the tree shrew is a viable new in vivo experimental model for avian influenza research that provides results comparable to those observed in ferrets. The disease spectrum and pathogenesis in tree shrews correlate well with what is observed in humans.

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID‐19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal‐type carbohydrates, mainly the N‐glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3‐galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase and α1,3‐galactosyl‐transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike receptor binding domain to produce glyco‐humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3‐galactose epitopes. Animals rapidly developed a hyperimmune response with anti‐SARS‐CoV‐2 end‐titers binding dilutions over one to a million and end‐titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV‐19, neutralized spike/angiotensin converting enzyme‐2 interaction at a concentration, GGTA1/CMAH double KO pigs produced high titers of XAV‐19, an anti‐SARS‐Cov‐2 RBD glyco‐humanized polyclonal antibody. XAV‐19 is presented with a high complement activation and a high neutralization potency in vitro and in cell‐based neutralization assays. XAV‐19 does not interact with human Fc gamma receptors, preventing Fc‐dependent enhancement or immune cells skewing.

SUMMARYAntigenic imprinting, which describes the bias of antibody response due to previous immune history, can influence vaccine effectiveness and has been reported in different viruses. Give that COVID-19 vaccine development is currently a major focus of the world, there is a lack of understanding of how background immunity influence antibody response to SARS-CoV-2. This study provides evidence for antigenic imprinting in Sarbecovirus, which is the subgenus that SARS-CoV-2 belongs to. Specifically, we sequentially immunized mice with two antigenically distinct Sarbecovirus strains, namely SARS-CoV and SARS-CoV-2. We found that the neutralizing antibodies triggered by the sequentially immunization are dominantly against the one that is used for priming. Given that the impact of the background immunity on COVID-19 is still unclear, our results will provide important insights into the pathogenesis of this disease as well as COVID-19 vaccination strategy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The SARS-CoV-2 virus emerged in December 2019 and has caused a worldwide pandemic due to the lack of any pre-existing immunity. Accurate serology testing is urgently needed to help diagnose infection, determine past exposure of populations and assess the response to a future vaccine. The landscape of antibody responses to SARS-CoV-2 is unknown. In this study, we utilized the luciferase immunoprecipitation system to assess the antibody responses to 15 different SARS-CoV-2 antigens in patients with COVID-19. We identified new targets of the immune response to SARS-CoV-2 and show that nucleocapsid, open reading frame (ORF)8 and ORF3b elicit the strongest specific antibody responses. ORF8 and ORF3b antibodies, taken together as a cluster of points, identified 96.5% of COVID-19 samples at early and late time points of disease with 99.5% specificity. Our findings could be used to develop second-generation diagnostic tests to improve serological assays for COVID-19 and are important in understanding pathogenicity.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory disease in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. We isolated the clade B MERS-CoV ChinaGD01 strain from a patient infected during the South Korean MERS outbreak in 2015 and compared the phylogenetics and pathogenicity of MERS-CoV EMC/2012 (clade A) and ChinaGD01 (clade B) in vitro and in vivo Genome alignment analysis showed that most clade-specific mutations occurred in the orf1ab gene, including mutations that were predicted to be potential glycosylation sites. Minor differences in viral growth but no significant differences in plaque size or sensitivity to beta interferon (IFN-β) were detected between these two viruses in vitro ChinaGD01 virus infection induced more weight loss and inflammatory cytokine production in human DPP4-transduced mice. Viral titers were higher in the lungs of ChinaGD01-infected mice than with EMC/2012 infection. Decreased virus-specific CD4+ and CD8+ T cell numbers were detected in the lungs of ChinaGD01-infected mice. In conclusion, MERS-CoV evolution induced changes to reshape its pathogenicity and virulence in vitro and in vivo and to evade adaptive immune response to hinder viral clearance.IMPORTANCE MERS-CoV is an important emerging pathogen and causes severe respiratory infection in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. In this study, we showed that a clade B virus ChinaGD01 strain caused more severe disease in mice, with delayed viral clearance, increased inflammatory cytokines, and decreased antiviral T cell responses, than the early clade A virus EMC/2012. Given the differences in pathogenicity of different clades of MERS-CoV, periodic assessment of currently circulating MERS-CoV is needed to monitor potential severity of zoonotic disease.

BackgroundSince early February 2021, the causative agent of COVID-19, SARS-CoV-2, has infected over 104 million people with more than 2 million deaths according to official reports. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown.MethodsUsing high-throughput sequencing of metatranscriptomic and hybrid captured libraries, we characterized consensus genomes and intra-host single nucleotide variations (iSNVs) of serial samples collected from eight patients with COVID-19. The distribution of iSNVs along the SARS-CoV-2 genome was analyzed and co-occurring iSNVs among COVID-19 patients were identified. We also compared the evolutionary dynamics of SARS-CoV-2 population in the respiratory tract (RT) and gastrointestinal tract (GIT).ResultsThe 32 consensus genomes revealed the co-existence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in a single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparing allele frequencies of the iSNVs revealed a clear genetic differentiation between intra-host populations from the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events during intra-host migrations. Compared to RT populations, the GIT populations showed a better maintenance and rapid development of viral genetic diversity following the suspected intra-host bottlenecks.ConclusionsOur findings here illustrate the intra-host bottlenecks and evolutionary dynamics of SARS-CoV-2 in different anatomic sites and may provide new insights to understand the virus-host interactions of coronaviruses and other RNA viruses.

Summary The World Health Organization has declared the ongoing outbreak of COVID-19, which is caused by a novel coronavirus SARS-CoV-2, as pandemic. There is currently a lack of knowledge about the antibody response elicited from SARS-CoV-2 infection. One major immunological question concerns antigenic differences between SARS-CoV-2 and SARS-CoV. We address this question by analyzing plasma from patients infected by SARS-CoV-2 or SARS-CoV, and from infected or immunized mice. Our results show that, while cross-reactivity in antibody binding to the spike protein is common, cross-neutralization of the live viruses may be rare, indicating the presence of non-neutralizing antibody response to conserved epitopes in the spike. Whether such low or non-neutralizing antibody response leads to antibody-dependent disease enhancement needs to be addressed in the future. Overall, this study not only addresses a fundamental question regarding antigenicity differences between SARS-CoV-2 and SARS-CoV, but also has implications for immunogen design and vaccine development., Graphical Abstract

As of middle May 2020, the causative agent of COVID-19, SARS-CoV-2, has infected over 4 million people with more than 300 thousand death as official reports1,2. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. Here, using deep sequencing data, we achieved and characterized consensus genomes and intra-host genomic variants from 32 serial samples collected from eight patients with COVID-19. The 32 consensus genomes revealed the coexistence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparison of allele frequencies of the iSNVs revealed genetic divergence between intra-host populations of the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events among intra-host transmissions. Nonetheless, we observed a maintained viral genetic diversity within GIT, showing an increased population with accumulated mutations developed in the tissue-specific environments. The iSNVs identified here not only show spatial divergence of intra-host viral populations, but also provide new insights into the complex virus-host interactions.

Background: The SARS-CoV-2 virus emerged in December 2019 and caused a pandemic associated with a spectrum of COVID-19 disease ranging from asymptomatic to lethal infection. Serology testing is important for diagnosis of infection, determining infection attack rates and immunity in the population. It also informs vaccine development. Although several serology tests are in use, improving their specificity and sensitivity for early diagnosis on the one hand and for detecting past infection for population-based studies, are priorities. Methods: We evaluated the anti-SARS-CoV-2 antibody profiles to 15 SARS-CoV-2 antigens by cloning and expressing 15 open reading frames (ORFs) in mammalian cells and screened antibody responses to them in COVID-19 patients using the Luciferase Immunoprecipitation System (LIPS). Results: The LIPS technique allowed us to detect antibody responses in COVID-19 patients to 11 of the 15 SARS-CoV-2 antigens tested, identifying novel immunogenic targets. This technique shows that antigens ORF3b and ORF8 allow detection of antibody early in infection in a specific manner and reveals the immuno-dominance of the N antigen in COVID-19 patients. Conclusion: Our report provides an unbiased characterization of antibody responses to a range of SARS-CoV-2 antigens. The combination of 3 SARS-CoV-2 antibody LIPS assays, i.e. N, ORF3b, and ORF8, is sufficient to identify all COVID-19 patients of our cohort even at early time-points of illness, whilst Spike alone fails to do so. Furthermore, our study highlights the importance of investigating new immunogens NSP1, ORF3b, ORF7a and ORF8 which may mediate immune functions other than neutralization which may be beneficial or harmful to the patient.

Targeting the SARS-CoV-2 spike The surface of severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) is decorated with trimeric spikes that bind to host cell receptors. These spikes also elicit an antibody response, so understanding antibody recognition may aid in vaccine design. Yuan et al. determined the structure of CR3022, a neutralizing antibody obtained from a convalescent SARS-CoV–infected patient, in complex with the receptor-binding domain of the SARS-CoV-2 spike. The antibody binds to an epitope conserved between SARS-CoV-2 and SARS-CoV that is distinct from the receptor-binding site. CR3022 likely binds more tightly to SARS-CoV because its epitope contains a glycan not present in SARS-CoV-2. Structural modeling showed that the epitope is only revealed when at least two of the three spike proteins are in a conformation competent to bind the receptor. Science , this issue p. 630

BackgroundHuman infection by avian influenza viruses is characterized by rapid development of acute respiratory distress and severe pneumonia. However, the underlying host response leading to this severe outcome is not well studied.MethodsWe conducted mass spectrometry-based serum proteome profiling on 10 healthy controls and 15 H7N9 infected cases with two time points and carried out statistical and biology functional enrichment analysis.ResultsIn total, we identified 647 proteins, 273 proteins were only found in H7N9 infected cases which might generate from cell leakage/death (apoptosis and/or necrosis) and identified 50 proteins with statistically significant difference between healthy control and H7N9 infected cases from 168 qualified proteins. We also found that M1 and PB2 tightly associated with the host’s HSPA8 (P11142, p=0.0042) which plays an important role in the protein quality control system.ConclusionsH7N9 infection may increase cell programmed/unprogrammed cell death, and we suggested that upregulated extracellular HSPA8 may suppress the H7N9 virion replication via activation amyloid-beta binding network.

Background The ongoing coronavirus disease (COVID-19) pandemic has major impacts on health systems, the economy and society. Assessing infection attack rates in the population is critical for estimating disease severity and herd immunity which is needed to calibrate public health interventions. We have previously shown that it is possible to achieve this in real time to impact public health decision making. Aim Our objective was to develop and evaluate serological assays applicable in large-scale sero-epidemiological studies. Methods We developed an ELISA to detect IgG and IgM antibodies to the receptor-binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated its sensitivity and specificity in combination with confirmatory microneutralisation (MN) and 90% plaque reduction neutralisation tests (PRNT90) in 51 sera from 24 patients with virologically confirmed COVID-19 and in age-stratified sera from 200 healthy controls. Results IgG and IgM RBD ELISA, MN and PRNT90 were reliably positive after 29 days from illness onset with no detectable cross-reactivity in age-stratified controls. We found that PRNT90 tests were more sensitive in detecting antibody than MN tests carried out with the conventional 100 tissue culture infectious dose challenge. Heparinised plasma appeared to reduce the infectivity of the virus challenge dose and may confound interpretation of neutralisation test. Conclusion Using IgG ELISA based on the RBD of the spike protein to screen sera for SARS-CoV-2 antibody, followed by confirmation using PRNT90, is a valid approach for large-scale sero-epidemiology studies.

This study correlated serial blood transcriptomic profiles of patients with avian influenza A (H7N9) virus infection with clinical data of patients. Biologically significant transcriptomic profiles associated with blood oxygenation and viral load in the lower respiratory tract were defined., Background Avian influenza A (H7N9) viruses emerged in China in 2013 and caused zoonotic disease associated with a case-fatality ratio of over 30%. Transcriptional profiles in peripheral blood reflect host responses and can help to elucidate disease pathogenesis. Methods We correlated serial blood transcriptomic profiles of patients with avian influenza A (H7N9) virus infection and determined the biological significances from the analysis. Results We found that specific gene expression profiles in the blood were strongly correlated with the Pao 2/Fio 2 ratio and viral load in the lower respiratory tract. Cell cycle and leukocyte-related immunity were activated at the acute stage of the infection while T-cell functions and various metabolic processes were associated with the recovery phase of the illness. A transition from systemic innate to adaptive immunity was found. Conclusions We developed a novel approach for transcriptomic analysis to identify key host responses that were strongly correlated with specific clinical and virologic parameters in patients with H7N9 infection.

Individuals with the 2019 coronavirus disease (COVID-19) show varying severity of the disease, ranging from asymptomatic to requiring intensive care. Although monoclonal antibodies specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been identified, we still lack an understanding of the overall landscape of B cell receptor (BCR) repertoires in individuals with COVID-19. We use high-throughput sequencing of bulk and plasma B cells collected at multiple time points during infection to characterize signatures of the B cell response to SARS-CoV-2 in 19 individuals. Using principled statistical approaches, we associate differential features of BCRs with different disease severity. We identify 38 significantly expanded clonal lineages shared among individuals as candidates for responses specific to SARS-CoV-2. Using single-cell sequencing, we verify the reactivity of BCRs shared among individuals to SARS-CoV-2 epitopes. Moreover, we identify the natural emergence of a BCR with cross-reactivity to SARS-CoV-1 and SARS-CoV-2 in some individuals. Our results provide insights important for development of rational therapies and vaccines against COVID-19., Graphical abstract, It is unclear how the dynamics of the humoral immune response to SARS-CoV-2 vary across individuals with different disease severity. Montague et al. develop a principled statistical approach based on time-course, high-throughput B cell repertoire sequences to identify shared, expanding, rare clonal B cell lineages as candidates for responses specific to SARS-CoV-2.

Mycoplasma pneumoniae is a common atypical respiratory pathogen causing community-acquired pneumonia in children. Co-infection with other respiratory viruses is common in pediatric patients but super-infection with bacteria other than Streptococcus pneumoniae and Haemophilus influenzae is rare. The first case of Chromobacterium violaceum infection incubated during and manifested after pneumonia caused by Mycoplasma pneumoniae in a 12-month old girl without any known history of immunodeficiency is here reported. The patient developed fever with redness and swelling over the middle phalanx of the right hand index finger which progressed to the formation of skin abscess. Following a course of intravenous meropenem and surgical drainage of the skin abscess, the patient fully recovered and was discharged.

H9N2 viruses are the most widespread influenza viruses in poultry in Asia. We evaluated the infection and tropism of human and avian H9 influenza virus in the human respiratory tract using ex vivo respiratory organ culture. H9 viruses infected the upper and lower respiratory tract and the majority of H9 viruses had a decreased ability to release virus from the bronchus rather than the lung. This may be attributed to a weak neuraminidase (NA) cleavage of carbon-6-linked sialic acid (Sia) rather than carbon-3-linked Sia. The modified cleavage of N-acetlylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) by NA in H9 virus replication was observed by reverse genetics, and recombinant H9N2 viruses with amino acids (38KQ) deleted in the NA stalk, and changing the amino acid at position 431 from Proline-to-Lysine. Using recombinant H9 viruses previously evaluated in the ferret, we found that viruses which replicated well in the ferret did not replicate to the same extent in the human ex vivo cultures. The existing risk assessment models for H9N2 viruses in ferrets may not always have a strong correlation with the replication in the human upper respiratory tract. The inclusion of the human ex vivo cultures would further strengthen the future risk-assessment strategies.

A novel avian-origin influenza A (H7N9) virus emerged in China in 2013 and has caused zoonotic disease in over 1123 persons with an overall mortality around 30%. Amino acid changes at the residues 591, 627 and 701 of polymerase basic protein 2 (PB2) have been found frequently in the human H7N9 isolates but not in viruses isolated from avian species. We have recently identified a cluster of H7N9 viruses in ducks which circulated in China prior to the first recognition of zoonotic disease in 2013. These duck viruses have genetic background distinct from the zoonotic H7N9 lineage. We found that the introduction of PB2 mutation with K at 627 but not K at 591 or N at 701 to the duck H7N9 virus led to increased pathogenicity in mice. We also found that the induction of pro-inflammatory cytokines including TNF-α, IP-10, MCP-1 and MIP-1α were associated with increased severity of infection. We conclude that introduction of the mammalian adaptation mutations into the PB2 gene of duck H7N9 viruses, which are genetically unrelated to the zoonotic H7N9 lineage, can also enhance pathogenicity in mice.

To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G1 phase. M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into S phase, failed to normalize virus production. Infection by influenza A virus triggered redistribution of cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. When overexpressed in HEK 293T cells, cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection.

The acute respiratory distress syndrome (ARDS) is the leading cause of death in influenza A virus (IAV)-infected patients. Hereby, the cellular importin-α7 gene plays a major role. It promotes viral replication in the lung, thereby increasing the risk for the development of pneumonia complicated by ARDS. Herein, we analyzed whether the recently emerged H7N9 avian IAV has already adapted to human importin-α7 use, which is associated with high-level virus replication in the mammalian lung. Using a cell-based viral polymerase activity assay, we could detect a decreased H7N9 IAV polymerase activity when importin-α7 was silenced by siRNA. Moreover, virus replication was diminished in the murine cells lacking the importin-α7 gene. Consistently, importin-α7 knockout mice presented reduced pulmonary virus titers and lung lesions as well as enhanced survival rates compared to wild-type mice. In summary, our results show that H7N9 IAV have acquired distinct features of adaptation to human host factors that enable enhanced virulence in mammals. In particular, adaptation to human importin-α7 mediates elevated virus replication in the mammalian lung, which might have contributed to ARDS observed in H7N9-infected patients.

Ethnopharmacological relevance Bai Shao (Radix Paeoniae Alba, BS), the root of Paeonia lactiflora Pall., in ancient China was used for Wen Bing (Warm Disease) treatment. Wen Bing has the symptoms of influenza. Ethanol extract of the root has recently been shown to possess anti-influenza activity. However, the active compounds have not yet been identified. Aim We showed that BS aqueous extract was potent in inhibiting influenza A virus in infected cells. We aimed to isolate the bioactive compounds and characterize the anti-influenza mechanism. Materials and methods Plaque reduction assay was performed for fractions isolated from BS. Hemagglutination inhibition assay and neuraminidase inhibition assay were performed to find the target protein. Molecular docking and reverse genetics were used to confirm the action site of gallic acid on the neuraminidase protein. Results We identified three tannin compounds gallic acid (GA), methyl gallate (MG) and pentagalloylglucose (PGG) in BS aqueous extract that could inhibit the replication of influenza A virus in MDCK cells. While only PGG was found to inhibit the influenza virus-induced hemagglutination of chicken erythrocytes, all three compounds significantly reduced the activity of the neuraminidase. The results from molecular docking and reverse genetics showed that GA interacted with Arg152 of neuraminidase protein. Conclusion Three compounds GA, MG and PGG isolated from BS were found to inhibit influenza A virus in MDCK cells. GA interacts with amino acid Arg152 of the viral neuraminidase. Our study identified anti-influenza compounds of BS and demonstrated their antiviral mechanism, thus providing scientific evidence for using this herb for clinical treatment.

SummaryDeubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host innate immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, DUBs that regulate this pathway have not been identified. Using a ubiquitin C-terminal electrophile, we profiled DUBs that function during influenza A virus (IAV) infection, and isolated OTUB1 as a key regulator of RIG-I dependent antiviral responses. OTUB1 was interferon-inducible, and interacted with RIG-I, viral PB2 and NS1. Upon infection, OTUB1 relocalised from the nucleus to mitochondrial membranes, and activated the RIG-I signaling complex via hydrolysis of K48 polyubiquitin chains and by forming a repressive complex with UBCH5c. Using a reconstituted system composed of in vitro translated [35S]IRF3, purified RIG-I, mitochondrial membranes and cytosol expressing OTUB1 variants, we recapitulated the mechanism of OTUB1-dependent RIG-I activation. A wide range of IAV NS1 proteins triggered proteasomal degradation of OTUB1, thereby antagonizing the RIG-I signaling cascade and antiviral responses.HighlightsOTUB1 is induced during influenza A virus infections in an IFN-I dependent mannerOTUB1 regulates the RIG-I complex by hydrolysing K48-linked polyubiquitin chains and by sequestering UBCH5c to prevent K48 polyubiquitylationOptimal K63 versus K48 polyubiquitin chain concentrations determine RIG-I activationInfluenza NS1 targets OTUB1 for proteasomal degradation

The influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization signal resides at the extended C-terminal region, spanning from R513 to K549. Our experimental data determined that the KKMK motif within this region plays important roles in both nuclear import and polymerase activity. Similar to the influenza A viruses, NPC also binds to multiple human importin α isoforms. Taken together, our results enhance the understanding of the virus-host interaction of the influenza C virus. IMPORTANCE As a member of the Orthomyxoviridae family, the polymerase complex of the influenza C virus structurally resembles its influenza A and influenza B virus counterparts, but the nucleoprotein differs by possessing an extra C-terminal region. We have characterized this region in view of nuclear import and interaction with the importin α protein family. Our results demonstrate the functional significance of a previously uncharacterized region on Orthomyxoviridae nucleoprotein (NP). Based on this work, we propose that importin α binding to influenza C virus NP is regulated by a long bipartite nuclear localization signal. Since the sequence of influenza D virus NP shares high homology to that of the influenza C virus, this work will also shed light on how influenza D virus NP functions.

To the Editor: Since the first detection of the influenza A(H5N1) virus in geese in China during 1996 (http://www.cdc.gov/flu/avianflu/h5n1-virus.htm), H5 subtype viruses have continued to reassort and evolve, giving rise to multiple virus clades and gene constellations. Recently, clade 2.3.4.4 viruses have shown a predilection for genetic reassortment, giving rise to H5N2, H5N5, H5N6, and H5N8 virus subtypes, and have become globally widespread, causing infections in wild birds or poultry elsewhere in Asia, and in Europe and North America (1–3). The H5N6 subtype viruses have circulated in China since 2013 and have been mainly identified in ducks or chickens in the southern (Jiangxi, Guangdong) or western (Sichuan) areas (4,5). Two lineages of H5N6 viruses with distant genetic background were found among the H5N6 viruses isolated in China (5). In China, there have been 3 cases of H5N6 virus infection among humans, causing 2 deaths. We recently reported the clinical characteristics and progression of a patient infected by the H5N6 virus in Guangzhou City, China, who was the second reported case-patient infected with this subtype (6). After having contact with poultry, he began to manifest an influenza-like illness on December 3, 2014, and progressed to a primary viral pneumonia. The H5N6 virus A/Guangzhou/39715/2014 (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"KP765785-KP765792","start_term":"KP765785","end_term":"KP765792","start_term_id":"766561162","end_term_id":"766561178"}}KP765785-KP765792) was isolated from a throat swab specimen collected on day 8 of his illness by inoculation into 9–11-day-old, specific pathogen-free embryonated chicken eggs. He recovered from his infection and was discharged from the hospital on day 58. Multiple sequence alignments showed that the hemagglutinin (HA) and neuraminidase (NA) genes of A/Guangzhou/39715/2014 shared the highest nucleotide identity with A/chicken/Dongguan/2690/2013 (H5N6) (99.4% and 98.3%, respectively) (Technical Appendix 1). All internal genes were also closely related to A/chicken/Dongguan/2690/2013 (H5N6), ranging from 98.5% nucleotide identity for the polymerase acidic (PA) gene and 100.0% for the matrix (M) gene. The genome segments were also 98.2%–99.7% identical to A/duck/Guangdong/{"type":"entrez-nucleotide","attrs":{"text":"GD012014","term_id":"198345769","term_text":"GD012014"}}GD012014 and 98.3%–99.4% identical to A/chicken/Laos/LPQ001/2014, which caused outbreaks in domestic ducks and poultry, respectively, indicating that these viruses have the same genotype. HA gene phylogeny confirmed that this virus belonged to clade 2.3.4.4 (Technical Appendix 1, Figure 4). Notably, the HA genes of the H5N1, H5N2, and H5N8 viruses that were recently detected in wild birds in North America also belong to this clade, indicating that viruses from this clade are becoming globally widespread. More specifically, this isolate clustered within a sublineage that includes H5N6 isolates from poultry from Guangdong and Jiangxi provinces, China, and from Laos (5,7). The A/Sichuan/26221/2014 (H5N6) virus that recently caused a fatal human infection in Sichuan Province, China is also within clade 2.3.4.4, but clusters in a distinct sub-lineage (Figure, panel A). Figure Phylogenetic trees of influenza A(H5N6) virus isolate A/Guangzhou/39715/2014 compared with other influenza viruses based on the A) hemagglutinin (HA) and B) neuraminidase (NA) genes, China. Maximum-likelihood trees were constructed by using the the general ... The HA cleavage site of both human isolates contained multiple basic amino acids, suggesting that that they are highly pathogenic avian influenza viruses. Amino acid substitutions E190D, Q226L, or G228S (H3 numbering) in the HA gene that are known to enhance binding to mammalian receptors were not found. The NA gene phylogeny showed that A/Guangzhou/39715/2014 is likely originated from group II lineage influenza A(H6N6) viruses that circulate among domestic ducks in China (8) (Figure, panel B). An 11-aa deletion at the residue 59–69 position of the NA protein was identified in the isolate of this study, in the other H5N6 viruses of the same cluster, and in an H4N6 virus isolate from a duck in Shanghai, China. This deletion was monophyletic and likely originated from A/swine/Guangdong/K6/2010 (H6N6)–like viruses (Figure, panel B). However, it was not observed in other 2.3.4.4 viruses, such as A/Sichuan/26221/2014. No mutations associated with oseltamivir or amantadine resistance was found in NA or M2 genes. The internal genes of the current H5N6 isolate were similar to 2.3.2.1b H5N1 subtype viruses found in domestic ducks from south-central and eastern China (5,7–10 [Technical Appendix 1]). The 6 internal genes are 97%–99% homologous to another isolate from a human, A/Sichuan/26221/2014, suggesting that the internal genes of the viruses may be reassorted from a common origin. The phylogenetic clustering observed for the HA gene was also conserved for the internal genes. In contrast with all avian viruses within this clade, the current human isolate contains the mammalian adaptation mutation PB2-E627K, and A/Sichuan/26221/2014 has acquired PB2-D701N, suggesting a rapid acquisition of mammalian adaptation changes that likely arose after human infection. There is still limited information on human disease caused by the emerging H5 lineage. Our genetic analysis suggests that the H5N6 virus isolated from the patient is originated from the avian host. Although the genetic background of H5N6 virus isolated from the third case in Yunnan Province, China, on January 2015 is still not known, the isolates from the human cases of H5N6 infection reported to date show distant genetic diversity, indicating that viruses from both clusters may pose a threat to humans. This rapidly evolving and globally spreading virus lineage thus provides a threat to global public health. Technical Appendix 1: Full phylogenetic trees of polymerase basic 2 (PB2) (Technical Appendix Figure 1), PB1 (Technical Appendix Figure 2), polymerase acidic (PA) (Technical Appendix Figure 3), hemagglutinin (HA) (Technical Appendix Figure 4), nucleoprotein (NP) (Technical Appendix Figure 5), neuraminidase (NA) (Technical Appendix Figure 6), matrix (M) (Technical Appendix Figure 7), and nonstructural (NS) (Technical Appendix Figure 8) genes. Click here to view.(2.1M, pdf) Technical Appendix 2: Authors and originating and submitting laboratories of the sequences from the Global Initiative on Sharing Avian Influenza Data EpiFlu Database, on which the current research is based. Click here to view.(63K, xlsx)

Influenza pandemic is a constant major threat to public health caused by influenza A viruses (IAV). IAVs are sub-categorized by the surface proteins: hemagglutinin (HA) and neuraminidase (NA), in which they are both essential targets for drug discovery. While it is of great concern that NA inhibitor oseltamivir resistant strains are frequently identified from human or avian influenza virus, structural and functional characterization of influenza HA has raised hopes for new antiviral therapies. In this study, we explored a structure-activity relationship (SAR) of pinanamine-based antivirals and discovered a potent inhibitor M090 against amantadine-resistant viruses, including the 2009 H1N1 pandemic strains, and oseltamivir- resistant viruses. Mechanism of action studies, particularly hemolysis inhibition, indicated that M090 targets influenza HA and it occupied a highly conserved pocket of the HA(2) domain and inhibited virus-mediated membrane fusion by “locking” the bending state of HA(2) during the conformational rearrangement process. This work provides new binding sites within the HA protein and indicates that this pocket may be a promising target for broad-spectrum anti-influenza A drug design and development.

Avian influenza A viruses are a threat to human health, as they cross the species barrier and infect humans occasionally, often with severe outcome. The antigenic and genetic diversity of A/H5 viruses from the A/goose/Guangdong/1/96 lineage is increasing, due to continued circulation and reassortment in poultry, posing a constant risk for public health and requiring regular risk assessments. Here we performed an in-depth characterization of the properties of the newly emerged zoonotic A/H5N6 virus in vitro and in ferrets. The lack of airborne transmission in the ferret model indicates that A/H5N6 virus does not pose a direct public health threat, despite the fact that it can replicate to high titers throughout the respiratory tracts of ferrets and cause more severe disease than other clade 2.3.4.4 viruses., Since their emergence in 1997, A/H5N1 influenza viruses of the A/goose/Guangdong/1/96 lineage have diversified in multiple genetic and antigenic clades upon continued circulation in poultry in several countries in Eurasia and Africa. Since 2009, reassortant viruses carrying clade 2.3.4.4 hemagglutinin (HA) and internal and neuraminidase (NA) genes of influenza A viruses of different avian origin have been detected, yielding various HA-NA combinations, such as A/H5N1, A/H5N2, A/H5N3, A/H5N5, A/H5N6, and A/H5N8. Previous studies reported on the low pathogenicity and lack of airborne transmission of A/H5N2 and A/H5N8 viruses in the ferret model. However, although A/H5N6 viruses are the only clade 2.3.4.4 viruses that crossed the species barrier and infected humans, the risk they pose for human health remains poorly characterized. Here, the characterization of A/H5N6 A/Guangzhou/39715/2014 virus in vitro and in ferrets is described. This A/H5N6 virus possessed high polymerase activity, mediated by the E627K substitution in the PB2 protein, which corresponds to only one biological trait out of the three that were previously shown to confer airborne transmissibility to A/H5N1 viruses between ferrets. This might explain its lack of airborne transmission between ferrets. After intranasal inoculation, A/H5N6 virus replicated to high titers in the respiratory tracts of ferrets and was excreted for at least 6 days. Moreover, A/H5N6 virus caused severe pneumonia in ferrets upon intratracheal inoculation. Thus, A/H5N6 virus causes a more severe disease in ferrets than previously investigated clade 2.3.4.4 viruses, but our results demonstrate that the risk from airborne spread is currently low. IMPORTANCE Avian influenza A viruses are a threat to human health, as they cross the species barrier and infect humans occasionally, often with severe outcome. The antigenic and genetic diversity of A/H5 viruses from the A/goose/Guangdong/1/96 lineage is increasing, due to continued circulation and reassortment in poultry, posing a constant risk for public health and requiring regular risk assessments. Here we performed an in-depth characterization of the properties of the newly emerged zoonotic A/H5N6 virus in vitro and in ferrets. The lack of airborne transmission in the ferret model indicates that A/H5N6 virus does not pose a direct public health threat, despite the fact that it can replicate to high titers throughout the respiratory tracts of ferrets and cause more severe disease than other clade 2.3.4.4 viruses.

Sequence analyses of influenza PB2 sequences indicate that the 627 position almost exclusively contains either lysine (K) or glutamic acid (E), suggesting a high sequence constraint at this genetic marker. Here, we used a site-directed random mutagenesis method to demonstrate that PB2-627 position has a high sequence plasticity. Recombinant viruses carrying various amino acid residues at this position are viable in cell cultures. These PB2-627 mutants showed various polymerase activities and replication kinetics in mammalian and avian cells as well as pathogenicity in mice. Serially passaging these mutants in MDCK cells generated some compensatory PB2 mutations that can restore polymerase activities of the PB2-627 mutants. Of these, PB2-D309N was identified as a novel one. Besides showing that influenza virus can tolerate a wide range of amino acid residues at the PB2-627 position, this study also demonstrates a potential strategy to identify novel mutations that can enhance viral polymerase.

We describe the epidemiology of highly pathogenic avian influenza (HPAI) A(H7N9) based on poultry market environmental surveillance and laboratory-confirmed human cases (n = 9) in Guangdong, China. We also compare the epidemiology between human cases of high- and low-pathogenic avian influenza A(H7N9) (n = 51) in Guangdong. Case fatality and severity were similar. Touching sick or dead poultry was the most important risk factor for HPAI A(H7N9) infections and should be highlighted for the control of future influenza A(H7N9) epidemics.

The recent increase in zoonotic avian influenza A(H7N9) disease in China is a cause of public health concern. Most of the A(H7N9) viruses previously reported have been of low pathogenicity. We report the fatal case of a patient in China who was infected with an A(H7N9) virus having a polybasic amino acid sequence at its hemagglutinin cleavage site (PEVPKRKRTAR/GL), a sequence suggestive of high pathogenicity in birds. Its neuraminidase also had R292K, an amino acid change known to be associated with neuraminidase inhibitor resistance. Both of these molecular features might have contributed to the patient's adverse clinical outcome. The patient had a history of exposure to sick and dying poultry, and his close contacts had no evidence of A(H7N9) disease, suggesting human-to-human transmission did not occur. Enhanced surveillance is needed to determine whether this highly pathogenic avian influenza A(H7N9) virus will continue to spread.

Toll-like receptors (TLRs) play an important role in innate immunity to virus infections. We investigated the role of TLR3 in the pathogenesis of H5N1 and pandemic H1N1 (pH1N1) influenza virus infections in mice. Wild-type mice and those defective in TLR3 were infected with influenza A/HK/486/97 (H5N1) or A/HK/415742/09 (pH1N1) virus. For comparison, mice defective in the gene for myeloid differential factor 88 (MyD88) were also infected with the viruses, because MyD88 signals through a TLR pathway different from TLR3. Survival and body weight loss were monitored for 14 days, and lung pathology, the lung immune-cell profile, viral load and cytokine responses were studied. H5N1-infected TLR3−/− mice had better survival than H5N1-infected WT mice, evident by significantly faster regain of body weight, lower viral titre in the lung and fewer pathological changes in the lung. However, this improved survival was not seen upon pH1N1 infection of TLR3−/− mice. In contrast, MyD88−/− mice had an increased viral titre and decreased leukocyte infiltration in the lungs after infection with H5N1 virus and poorer survival after pH1N1 infection. In conclusion, TLR3 worsens the pathogenesis of H5N1 infection but not of pH1N1 infection, highlighting the differences in the pathogenesis of these two viruses and the different roles of TLR3 in their pathogenesis.

Avian H7N9 influenza viruses may pose a further threat to humans by reassortment with human viruses, which could lead to generation of novel reassortants with enhanced polymerase activity. We previously established a novel statistical approach to study the polymerase activity of reassorted vRNPs (Influenza Other Respir Viruses. 2013;7:969-78). Here, we report the use of this method to study recombinant vRNPs with subunits derived from human H1N1, H3N2, and H7N9 viruses. Our results demonstrate that some reassortant vRNPs with subunits derived from the H7N9 and other human viruses can have much higher polymerase activities than the wild-type levels., published_or_final_version

Human disease caused by highly pathogenic avian influenza A (HPAI) (H5N1) is associated with fulminant viral pneumonia and mortality rates in excess of 60%. Acute respiratory syndrome (ARDS) has been found to be the most severe form of acute lung injury caused by H5N1 virus infection while cytokine dysregulation and viral replication are thought to contribute to its pathogenesis. In this study, the antiviral and anti-inflammatory effects of two indirubin derivatives: indirubin-3′-oxime (IM) and E804 on primary human peripherial blood-derived macrophages and type-I like pneumocytes (human alveolar epithelial cells) during influenza A (H5N1) virus infection were investigated. We found that both of the indirubin derivatives strongly suppress the pro-inflammatory cytokines including IP-10 (CXCL10), one of the key factors which contribute to the lung inflammation during H5N1 virus infection. In addition, we also demonstrated that the indirubin derivative delays the virus replication in the primary cell culture models. Our results showed that indirubin derivatives have a potential to be used as an adjunct to antiviral therapy for the treatment of severe human H5N1 disease.

Session - Medical Sciences, Current influenza vaccines are ineffective against novel viruses and the source or the strain of the next outbreak of influenza is unpredictable; therefore, establishing universal immunity by vaccination to limit the impact of influenza remains a high priority. To meet this challenge, a novel vaccine has been developed using the immunogenic live vaccinia virus as a vaccine vector, expressing multiple H5N1 viral proteins (HA, NA, M1, M2, and NP) together with IL-15 as a molecular adjuvant. Previously, this vaccine demonstrated robust sterile cross-clade protection in mice against H5 influenza viruses, and herein its use has been extended to mediate heterosubtypic immunity toward viruses from both group 1 and 2 HA lineages. The vaccine protected mice against lethal challenge by increasing survival and significantly reducing lung viral loads against the most recent human H7N9, seasonal H3N2, pandemic-2009 H1N1, and highly pathogenic H7N7 influenza A viruses. Influenza-specific antibodies elicited by the vaccine failed to neutralize heterologous viruses and were unable to confer protection by passive transfer. Importantly, heterologous influenza-specific CD4(+) and CD8(+) T-cell responses that were elicited by the vaccine were effectively recalled and amplified following viral challenge in the lungs and periphery. Selective depletion of T-cell subsets in the immunized mice revealed an important role for CD4(+) T cells in heterosubtypic protection, despite low sequence conservation among known MHC-II restricted epitopes across different influenza viruses. This study illustrates the potential utility of our multivalent Wyeth/IL-15/5Flu as a universal influenza vaccine with a correlate of protective immunity that is independent of neutralizing antibodies., link_to_OA_fulltext

Novel avian H7N9 virus emerged in China in 2013 resulting in a case fatality rate of around 39% and continues to pose zoonotic and pandemic risk. Amino acid substitutions in PB2 protein were shown to influence the pathogenicity and transmissibility of H7N9 following experimental infection of ferrets and mice. In this study, we evaluated the role of amino acid substitution PB2-627K or compensatory changes at PB2-591K and PB2-701N, on the tropism and replication competence of H7N9 viruses for human and swine respiratory tracts using ex vivo organ explant cultures. Recombinant viruses of A/Shanghai/2/2013 (rgH7N9) and its mutants with PB2-K627E, PB2-K627E + Q591K and PB2-K627E + D701N were generated by plasmid-based reverse genetics. PB2-E627K was essential for efficient replication of rgH7N9 in ex vivo cultures of human and swine respiratory tracts. Mutant rgPB2-K627E + D701N replicated better than rgPB2-K627E in human lung but not as well as rgH7N9 virus. The rgPB2-K627E mutant failed to replicate in human type I-like pneumocytes (ATI) and peripheral blood monocyte-derived macrophages (PMϕ) at 37 °C while the compensatory mutant rgPB2-K627E + Q591K and rgPB2-K627E + D701N had partly restored replication competence in PMϕ. Our results demonstrate that PB2-E627K was important for efficient replication of influenza H7N9 in both human and swine respiratory tracts.

Summary Egg-based seasonal influenza vaccines are the major preventive countermeasure against influenza virus. However, their effectiveness can be compromised when antigenic changes arise from egg-adaptive mutations on influenza hemagglutinin (HA). The L194P mutation is commonly observed in egg-based H3N2 vaccine seed strains and significantly alters HA antigenicity. An approach to prevent L194P would therefore be beneficial. We show that emergence of L194P during egg passaging can be impeded by preexistence of a G186V mutation, revealing strong incompatibility between these mutations. X-ray structures illustrate that individual G186V and L194P mutations have opposing effects on the HA receptor-binding site (RBS), and when both G186V and L194P are present, the RBS is severely disrupted. Importantly, wild-type HA antigenicity is maintained with G186V, but not L194P. Our results demonstrate that these epistatic interactions can be used to prevent the emergence of mutations that adversely alter antigenicity during egg adaptation., Graphical Abstract, Highlights • Most H3N2 egg isolates carry hemagglutinin mutation G186V or L194P, but not both • Hemagglutinin double mutation G186V/L194P is highly deleterious to the virus • Hemagglutinin double mutation G186V/L194P disrupts the receptor-binding site • Wild-type hemagglutinin antigenicity is maintained in G186V, but not in L194P, Wu et al. characterize the incompatibility between two major egg-adaptive mutations in human H3N2 virus hemagglutinin, G186V and L194P, which confer either minimal or significant antigenic change, respectively. This study suggests that the antigenically disruptive mutation L194P that occurs during egg-based influenza vaccine production can be prevented by mutational incompatibility.

Summary Background Since March, 2013, an avian-origin influenza A H7N9 virus has caused severe pneumonia in China. The aim of this study was to investigate the pathogenesis of this new virus in human beings. Methods We obtained ex-vivo cultures of the human bronchus, lung, nasopharynx, and tonsil and in-vitro cultures of primary human alveolar epithelial cells and peripheral blood monocyte-derived macrophages. We compared virus tropism and induction of proinflammatory cytokine responses of two human influenza A H7N9 virus isolates, A/Shanghai/1/2013 and A/Shanghai/2/2013; a highly pathogenic avian influenza H5N1 virus; the highly pathogenic avian influenza H7N7 virus that infected human beings in the Netherlands in 2003; the 2009 pandemic influenza H1N1 virus, and a low pathogenic duck H7N9 virus that was genetically different to the human disease causing A H7N9 viruses. Findings Both human H7N9 viruses replicated efficiently in human bronchus and lung ex-vivo cultures, whereas duck/H7N9 virus failed to replicate in either. Both human A H7N9 viruses infected both ciliated and non-ciliated human bronchial epithelial cells and replicated to higher titres than did H5N1 (p