Structural basis of human ACE2 higher binding affinity to currently circulating Omicron SARS-CoV-2 sub-variants BA.2 and BA.1.1.

The currently circulating Omicron sub-variants are the SARS-CoV-2 strains with the highest number of known mutations. Herein, we found that human angiotensin-converting enzyme 2 (hACE2) binding affinity to the receptor-binding domains (RBDs) of the four early Omicron sub-variants (BA.1, BA.1.1, BA.2, and BA.3) follows the order BA.1.1 > BA.2 > BA.3 ≈ BA.1. The complex structures of hACE2 with RBDs of BA.1.1, BA.2, and BA.3 reveal that the higher hACE2 binding affinity of BA.2 than BA.1 is related to the absence of the G496S mutation in BA.2. The R346K mutation in BA.1.1 majorly affects the interaction network in the BA.1.1 RBD/hACE2 interface through long-range alterations and contributes to the higher hACE2 affinity of the BA.1.1 RBD than the BA.1 RBD. These results reveal the structural basis for the distinct hACE2 binding patterns among BA.1.1, BA.2, and BA.3 RBDs.

The structural basis of African swine fever virus pA104R binding to DNA and its inhibition by stilbene derivatives.

African swine fever virus (ASFV) is a highly contagious nucleocytoplasmic large DNA virus (NCLDV) that causes nearly 100% mortality in swine. The development of effective vaccines and drugs against this virus is urgently needed. pA104R, an ASFV-derived histone-like protein, shares sequence and functional similarity with bacterial HU/IHF family members and is essential for viral replication. Herein, we solved the crystal structures of pA104R in its apo state as well as in complex with DNA. Apo-pA104R forms a homodimer and folds into an architecture conserved in bacterial heat-unstable nucleoid proteins/integration host factors (HUs/IHFs). The pA104R-DNA complex structure, however, uncovers that pA104R has a DNA binding pattern distinct from its bacterial homologs, that is, the ß-ribbon arms of pA104R stabilize DNA binding by contacting the major groove instead of the minor groove. Mutations of the basic residues at the base region of the ß-strand DNA binding region (BDR), rather than those in the ß-ribbon arms, completely abolished DNA binding, highlighting the major role of the BDR base in DNA binding. An overall DNA bending angle of 93.8° is observed in crystal packing of the pA104R-DNA complex structure, which is close to the DNA bending angle in the HU-DNA complex. Stilbene derivatives SD1 and SD4 were shown to disrupt the binding between pA104R and DNA and inhibit the replication of ASFV in primary porcine alveolar macrophages. Collectively, these results reveal the structural basis of pA104R binding to DNA highlighting the importance of the pA104R-DNA interaction in the ASFV replication cycle and provide inhibitor leads for ASFV chemotherapy.

Primary Pancreatic Neuroblastoma in an Infant.

A 2-month-old girl with conjugated hyperbilirubinemia was found at the surgery and by computed tomography to have a large mass originating in the pancreas. Histopathology, molecular testing, and staging evaluations showed this to be a stage 3, MYCN unamplified, intermediate-risk neuroblastoma. The patient had a partial response to risk-stratified chemotherapy. The mass remained unresectable, but the response was sustained after 18 months. Although fewer than a dozen cases of primary pancreatic neuroblastoma have been reported, our experience and a literature review suggest that these tumors can be managed in the same way as similar-risk neuroblastoma of other sites.

Bunyavirales ribonucleoproteins: the viral replication and transcription machinery.

The Bunyavirales order is one of the largest groups of segmented negative-sense single-stranded RNA viruses, which includes many pathogenic strains that cause severe human diseases. The RNA segments of the bunyavirus genome are separately encapsidated by multiple copies of nucleoprotein (N), and both termini of each N-encapsidated genomic RNA segment bind to one copy of the viral L polymerase protein. The viral genomic RNA, N and L protein together form the ribonucleoprotein (RNP) complex that constitutes the molecular machinery for viral genome replication and transcription. Recently, breakthroughs have been achieved in understanding the architecture of bunyavirus RNPs with the determination of the atomic structures of the N and L proteins from various members of this order. In this review, we discuss the structures and functions of these bunyavirus RNP components, as well as viral genome replication and transcription mechanisms.

Conserved epitopes dominate cross-CD8+ T-cell responses against influenza A H1N1 virus among Asian populations.

Novel strains of influenza A viruses (IAVs) have emerged with high infectivity and/or pathogenicity in recent years, causing worldwide concern. T cells are correlated with protection in humans through cross-reactive immunity against heterosubtypes of IAV. However, the different hierarchical roles of IAV-derived epitopes with distinct levels of polymorphism in the cross-reactive T-cell responses against IAV remain elusive. In this study, immunodominant epitopes scattered throughout the entire proteome of 2009 pandemic influenza A H1N1 virus and seasonal IAVs were synthesized and divided into different pools depending on their conservation. The overall profile of the IAV-specific CD8(+) T-cell immunity was detected by utilizing these peptide pools and also individual peptides. A dominant role of the conserved peptide-specific T-cell immunity was illuminated within the anti-IAV responses, while the CD8(+) T-cell responses against the variable epitopes were lower than the conserved peptides. As previously demonstrated within a Caucasian population, we determined that GL9-specific T cells, which also utilize Vß 17 TCR (BV19), play a pivotal role in IAV-specific T-cell immunity within an HLA-A2(+) Asian population. Our study objectively reveals the different predominant roles of T-cell epitopes among IAV-specific cross-reactive cellular immunity. This may guide the development of vaccines with cross-T-cell immunogenicity against heterosubtypes of IAV.

From endocytosis to membrane fusion: emerging roles of dynamin in virus entry.

Dynamin, a large guanosine triphosphatase (GTPase), has been implicated in virus entry, but its mechanisms of action are controversial. The entry procedure of most enveloped viruses involves endocytosis and membrane fusion. Dynamin has been suggested to act both as a regulatory GTPase by controlling the early stages of clathrin-mediated endocytosis (CME), which is an important endocytic pathway utilized by many viruses, and as a mechanochemical enzyme that induces membrane fission and pinches endocytic vesicles off from the cellular plasma membrane in later stages in several endocytic pathways, including CME. In addition to its involvement in virus endocytosis, dynamin has also been proposed to participate in membrane fusion between the virus and endosomes following endocytosis. Crystal structures and cryo-electron micrography (cryo-EM) have elucidated the structure of dynamin, which led to development of a mechanochemical model of how dynamin-mediated membrane fission occurs. Based on this, we propose a hypothetical model that explains how dynamin facilitates virus membrane fusion and discuss its roles in virus entry.

Characterization of RBD-specific cross-neutralizing antibodies responses against SARS-CoV-2 variants from COVID-19 convalescents.

Introduction: The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been posing a severe threat to global public health. Although broadly neutralizing antibodies have been used to prevent or treat corona virus disease 2019 (COVID-19), new emerging variants have been proven resistant to these antibodies. Methods: In this study, we isolated receptor binding domain (RBD)-specific memory B cells using single-cell sorting method from two COVID-19 convalescents and expressed the antibody to test their neutralizing activity against diverse SARS-CoV-2 variants. Then, we resolved antibody-RBD complex structures of potent RBD-specific neutralizing antibodies by X-ray diffraction method. Finally, we analyzed the whole antibody repertoires of the two donors and studied the evolutionary pathway of potent neutralizing antibodies. Results and discussion: We identified three potent RBD-specific neutralizing antibodies (1D7, 3G10 and 3C11) from two COVID-19 convalescents that neutralized authentic SARS-CoV-2 WH-1 and Delta variant, and one of them, 1D7, presented broadly neutralizing activity against WH-1, Beta, Gamma, Delta and Omicron authentic viruses. The resolved antibody-RBD complex structures of two antibodies, 3G10 and 3C11, indicate that both of them interact with the external subdomain of the RBD and that they belong to the RBD-1 and RBD-4 communities, respectively. From the antibody repertoire analysis, we found that the CDR3 frequencies of the light chain, which shared high degrees of amino acid identity with these three antibodies, were higher than those of the heavy chain. This research will contribute to the development of RBD-specific antibody-based drugs and immunogens against multiple variants.

Structural basis for receptor binding and broader interspecies receptor recognition of currently circulating Omicron sub-variants.

Multiple SARS-CoV-2 Omicron sub-variants, such as BA.2, BA.2.12.1, BA.4, and BA.5, emerge one after another. BA.5 has become the dominant strain worldwide. Additionally, BA.2.75 is significantly increasing in some countries. Exploring their receptor binding and interspecies transmission risk is urgently needed. Herein, we examine the binding capacities of human and other 28 animal ACE2 orthologs covering nine orders towards S proteins of these sub-variants. The binding affinities between hACE2 and these sub-variants remain in the range as that of previous variants of concerns (VOCs) or interests (VOIs). Notably, R493Q reverse mutation enhances the bindings towards ACE2s from humans and many animals closely related to human life, suggesting an increased risk of cross-species transmission. Structures of S/hACE2 or RBD/hACE2 complexes for these sub-variants and BA.2 S binding to ACE2 of mouse, rat or golden hamster are determined to reveal the molecular basis for receptor binding and broader interspecies recognition.

The membrane protein of severe acute respiratory syndrome coronavirus acts as a dominant immunogen revealed by a clustering region of novel functionally and structurally defined cytotoxic T-lymphocyte epitopes.

BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged with highly contagious and life-threatening characteristics in 2002, remains a potential risk for future outbreaks. Membrane (M) and envelope (E) proteins are major structural proteins of the SARS-CoV. The M protein has been determined as a protective antigen in humoral responses. However, its potential roles in stimulating cellular immunity remain elusive. METHODS: In this study, a panel of peptides derived from M and E proteins were tested by in vitro refolding, T2 cell-binding assays, and responses stimulated by cytotoxic T-lymphocyte (CTL) epitopes in HLA-A2.1/K(b) transgenic mice and human peripheral blood mononuclear cells (PBMCs). RESULTS: A nonameric epitope Mn2 and a decameric epitope Md3 derived from the M protein were identified and used for the evaluation of M protein-specific immunity. Responses stimulated by M protein-specific CTL epitopes have been found in the PBMCs of donors who had recovered from SARS infection. Additionally, the transmembrane domain of the M protein may contain a T cell epitope cluster revealed by the immunogenic and structural analysis of a panel of truncated peptides overlapping with Mn2 and Md3. CONCLUSIONS: The M protein of SARS-CoV holds dominant cellular immunogenicity. This, together with previous reports of a strong humoral response against the M protein, may help to further explain the immunogenicity of SARS and serves as potential targets for SARS-CoV vaccine design.

Computational predicting the human infectivity of H7N9 influenza viruses isolated from avian hosts.

The genome composition of a given avian influenza virus is the primary determinant of its potential for cross-species transmission from birds to humans. Here, we introduce a viral genome-based computational tool that can be used to evaluate the human infectivity of avian isolates of influenza A H7N9 viruses, which can enable prediction of the potential risk of these isolates infecting humans. This tool, which is based on a novel class weight-biased logistic regression (CWBLR) algorithm, uses the sequences of the eight genome segments of an H7N9 strain as the input and gives the probability of this strain infecting humans (reflecting its human infectivity). We examined the replication efficiency and the pathogenicity of several H7N9 avian isolates that were predicted to have very low or high human infectivity by the CWBLR model in cell culture and in mice, and found that the strains with high predicted human infectivity replicated more efficiently in mammalian cells and were more infective in mice than those that were predicted to have low human infectivity. These results demonstrate that our CWBLR model can serve as a powerful tool for predicting the human infectivity and cross-species transmission risks of H7N9 avian strains.

The molecular basis for SARS-CoV-2 binding to dog ACE2.

SARS-CoV-2 can infect many domestic animals, including dogs. Herein, we show that dog angiotensin-converting enzyme 2 (dACE2) can bind to the SARS-CoV-2 spike (S) protein receptor binding domain (RBD), and that both pseudotyped and authentic SARS-CoV-2 can infect dACE2-expressing cells. We solved the crystal structure of RBD in complex with dACE2 and found that the total number of contact residues, contact atoms, hydrogen bonds and salt bridges at the binding interface in this complex are slightly fewer than those in the complex of the RBD and human ACE2 (hACE2). This result is consistent with the fact that the binding affinity of RBD to dACE2 is lower than that of hACE2. We further show that a few important mutations in the RBD binding interface play a pivotal role in the binding affinity of RBD to both dACE2 and hACE2. Our work reveals a molecular basis for cross-species transmission and potential animal spread of SARS-CoV-2, and provides new clues to block the potential transmission chains of this virus.

Identification and structural definition of H5-specific CTL epitopes restricted by HLA-A*0201 derived from the H5N1 subtype of influenza A viruses.

The haemagglutinin (HA) glycoprotein of influenza A virus is a major antigen that initiates humoral immunity against infection; however, the cellular immune response against HA is poorly understood. Furthermore, HA-derived cytotoxic T-lymphocyte (CTL) epitopes are relatively rare in comparison to other internal gene products. Here, CTL epitopes of the HA serotype H5 protein were screened. By using in silico prediction, in vitro refolding and a T2 cell-binding assay, followed by immunization of HLA-A2.1/K(b) transgenic mice, an HLA-A*0201-restricted decameric epitope, RI-10 (H5 HA205-214, RLYQNPTTYI), was shown to elicit a robust CTL epitope-specific response. In addition, RI-10 and its variant, KI-10 (KLYQNPTTYI), were also demonstrated to be able to induce a higher CTL epitope-specific response than the influenza A virus dominant CTL epitope GL-9 (GILGFVFTL) in peripheral blood mononuclear cells of HLA-A*0201-positive patients who had recovered from H5N1 virus infection. Furthermore, the crystal structures of RI-10-HLA-A*0201 and KI-10-HLA-A*0201 complexes were determined at 2.3 and 2.2 A resolution, respectively, showing typical HLA-A*0201-restricted epitopes. The conformations of RI-10 and KI-10 in the antigen-presenting grooves in crystal structures of the two complexes show significant differences, despite their nearly identical sequences. These results provide implications for the discovery of diagnostic markers and the design of novel influenza vaccines.

A novel recombinant peptide containing only two T-cell tolerance epitopes of chicken type II collagen that suppresses collagen-induced arthritis.

Immunotherapy of rheumatoid arthritis (RA) using oral-dosed native chicken or bovine type II collagen (nCII) to induce specific immune tolerance is an attractive strategy. However, the majority of clinical trials of oral tolerance in human diseases including RA in recent years have been disappointing. Here, we describe a novel recombinant peptide rcCTE1-2 which contains only two tolerogenic epitopes (CTE1 and CTE2) of chicken type II collagen (cCII). These are the critical T-cell determinants for suppression of RA that were first developed and used to compare its suppressive effects with ncCII on the collagen-induced arthritis (CIA) model. The rcCTE1-2 was produced using the prokaryotic pET expression system and purified by Ni-NTA His affinity chromatography. Strikingly, our results showed clearly that rcCTE1-2 was as efficacious as ncCII at the dose of 50 microg/kg/d. This dose significantly reduced footpad swelling, arthritic incidence and scores, and deferred the onset of disease. Furthermore, rcCTE1-2 of 50 microg/kg/d could lower the level of anti-nCII antibody in the serum of CIA animals, decrease Th1-cytokine INF-gamma level, and increase Th3-cytokine TGF-beta(1) produced level by spleen cells from CIA mice after in vivo stimulation with ncCII. Importantly, rcCTE1-2 was even more potent than native cCII, which was used in the clinic for RA. Equally importantly, the findings that the major T-cell determinants of cCII that are also recognized by H-2(b) MHC-restricted T cells have not previously been reported. Taken together, these results suggest that we have successfully developed a novel recombinant peptide rcCTE1-2 that can induce a potent tolerogenic response in CIA.

Analysis of ACE2 Gene-Encoded Proteins Across Mammalian Species.

Human beings are currently experiencing a serious public health event. Novel coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has infected about 3 million people worldwide and killed more than 200,000, most being the elderly or people with potential chronic diseases or in immunosuppressive states. According to big data analysis, there are many proteins homologous to or interacting with the angiotensin-converting enzyme 2 (ACE2), which, therefore, may not be the only receptor for the novel coronavirus; other receptors may also exist in host cells of different species. These potential receptors may also play an important role in the infection process of the novel coronavirus. The current study aimed to discover such key proteins or receptors and analyze the susceptibility of different animals to the novel coronavirus, in order to reveal the transmission process of the virus in cross-species infection. We analyzed the proteins coded by the ACE2 gene in different mammalian species and predicted their correlation and homology with the human ACE2 receptor. The major finding of our predictive analysis suggested ACE2 gene-encoded proteins to be highly homologous across mammals. Based on their high homology, their possibility of binding the spike-protein of SARS-CoV-2 is quite high and species such as Felis catus, Bos taurus, Rattus norvegicus etc. may be potential susceptible hosts; special monitoring is particularly required for livestock that are in close contact with humans. Our results might provide ideas for the prevention and control of the novel coronavirus pneumonia.

Comparison of Long-term Survival of Patients With Early-Stage Non-Small Cell Lung Cancer After Surgery vs Stereotactic Body Radiotherapy.

Importance: Previous comparisons of surgery and stereotactic body radiotherapy (SBRT) for early-stage (ES) non-small cell lung cancer (NSCLC) did not account for the extent of regional lymph node examination (LNE) during surgery. Objective: To compare long-term overall survival (OS) of patients with ES NSCLC after surgery vs SBRT when the extent of regional LNE in patients undergoing surgery is thoroughly considered. Design, Setting, and Participants: Cohort study with survival comparisons using the multivariable Cox proportional hazards model and after propensity score matching. Data from the National Cancer Database were analyzed from October 28, 2018, through April 18, 2019. Patients with ES NSCLC diagnosed between January 1, 2004, and December 31, 2015, who underwent any curative-intent surgery or SBRT were included. Main Outcomes and Measures: Long-term OS. Results: Of 104 709 total patients, 91 330 underwent surgery (42 508 [46.5%] male; median [interquartile range] age, 68 [61-75] years) and 13 379 received SBRT (6065 [45.3%] male; median [interquartile range] age, 75 [68-81] years). Surgery, especially lobectomy (hazard ratio [HR], 0.53; 95% CI, 0.50-0.56), and regional LNE, especially when more than 10 lymph nodes were examined (HR, 0.73; 95% CI, 0.69-0.77), were associated with better long-term OS (P < .001). Pneumonectomy was not associated with reduced mortality risk when 0 nodes were examined (HR for stage T1, 1.43; 95% CI, 0.67-3.06; P = .35; HR for stage T2-T3, 0.62; 95% CI, 0.34-1.13; P = .12) or when more than 15 nodes were examined for stage T1 disease in patients younger than 80 years (HR, 0.77; 95% CI, 0.54-1.09; P = .14) or when patients aged 80 years or older received regional LNE of any extent (>15 nodes examined: HR for stage T1, 0.65; 95% CI, 0.16-2.64; P = .54; HR for stage T2-T3, 0.90; 95% CI, 0.50-1.60; P = .71). Less extensive surgery was not associated with improved OS when 0 nodes were examined in patients aged 80 years or older with stage T2 to T3 tumors (HR for lobectomy, 0.90; 95% CI, 0.65-1.25; P = .53) and in selected operable patients older than 75 years with stage T1 tumors (HR for lobectomy, 1.07; 95% CI, 0.57-2.00; P = .84). Conclusions and Relevance: This study found that, overall, surgery coupled with regional LNE of appropriate extent was associated with the best long-term OS in patients with ES NSCLC.

Crystal Structure of African Swine Fever Virus dUTPase Reveals a Potential Drug Target.

E165R, a highly specific dUTP nucleotidohydrolase (dUTPase) encoded by the African swine fever virus (ASFV) genome, is required for productive replication of ASFV in swine macrophages. Here, we solved the high-resolution crystal structures of E165R in its apo state and in complex with its product dUMP. Structural analysis explicitly defined the architecture of the active site of the enzyme as well as the interaction between the active site and the dUMP ligand. By comparing the ASFV E165R structure with dUTPase structures from other species, we found that the active site of E165R is highly similar to those of dUTPases from Mycobacterium tuberculosis and Plasmodium falciparum, against which small-molecule chemicals have been developed, which could be the potential drug or lead compound candidates for ASFV. Our results provide important basis for anti-ASFV drug design by targeting E165R.IMPORTANCE African swine fever virus (ASFV), an Asfivirus affecting pigs and wild boars with up to 100% case fatality rate, is currently rampaging throughout China and some other countries in Asia. There is an urgent need to develop therapeutic and preventive reagents against the virus. Our crystallographic and biochemical studies reveal that ASFV E165R is a member of trimeric dUTP nucleotidohydrolase (dUTPase) family that catalyzes the hydrolysis of dUTP into dUMP. Our apo-E165R and E165R-dUMP structures reveal the constitutive residues and the configuration of the active center of this enzyme in rich detail and give evidence that the active center of E165R is very similar to that of dUTPases from Plasmodium falciparum and Mycobacterium tuberculosis, which have already been used as targets for designing drugs. Therefore, our high-resolution structures of E165R provide useful structural information for chemotherapeutic drug design.

Cryo-EM Structure of the African Swine Fever Virus.

African swine fever virus (ASFV) is a large double-stranded DNA virus with an icosahedral multilayered structure. ASFV causes a lethal swine hemorrhagic disease and is currently responsible for widespread damage to the pork industry in Asia. Neither vaccines nor antivirals are available and the molecular characterization of the ASFV particle is outstanding. Here, we describe the cryogenic electron microscopy (cryo-EM) structure of the icosahedral capsid of ASFV at 4.6-Å. The ASFV particle consists of 8,280 copies of the major capsid protein p72, 60 copies of the penton protein, and at least 8,340 minor capsid proteins, of which there might be 3 different types. Like other nucleocytoplasmic large DNA viruses, the minor capsid proteins form a hexagonal network below the outer capsid shell, functioning as stabilizers by "gluing" neighboring capsomers together. Our findings provide a comprehensive molecular model of the ASFV capsid architecture that will contribute to the future development of countermeasures, including vaccines.

[Research advance of influenza B viruses].

Influenza B virus (IBV) is a segmented negative-strand RNA virus, which often causes local outbreak or seasonal epidemic along with influenza A virus (IAV) in the world. It is pathogenic to children, teenagers and elderly people and has a higher mortality rate in children and adolescents, so it poses a serious threat to public health and health. IBV is more likely to cause complications than IAV and the disease burden of IBV even exceeds IAV in the epidemic season. Recently, especially after winter of 2017, IBV has become the dominant strain in many areas of our country and seriously affects people's health. In view of this, this article reviews the structure, epidemiology, immunology and prevention of IBV, aiming at enhancing public's perceptions of the virus and providing reference for making strategies for prevention and control of influenza B.

The Hepatitis B Surface Antigen Binding Protein: An Immunoglobulin G Constant Region-Like Protein That Interacts With HBV Envelop Proteins and Mediates HBV Entry.

Hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis, liver cancer, and liver failure, affecting 350 million people worldwide. Currently available anti-HBV drugs include (PEGylated-) interferon-α and nucleos(t)ide analogs, which can cause significant side effects and drug-resistance in many cases of long-term treatment. The lack of a reliable and robust in vitro infection system is a major barrier for understanding the HBV life cycle and discovering novel therapeutic targets. In the present study, we demonstrate that overexpression of the hepatitis B surface antigen binding protein (SBP) in HepG2 cells (HepG2-SBP) resulted in their susceptibility to HBV infection. HepG2-SBP cells supported the uptake of the viral surface protein (HBsAg-preS), HBV-pseudotyped virus, and live HBV in patient sera. Moreover, SBP-mediated HBsAg-preS uptake, and HBV pseudotyped virus infections were efficiently blocked by preS1- and SBP-specific antibodies. These observations suggest that SBP is involved in HBV entry and that HepG2-SBP cells can serve as a cellular model to study the post-binding steps of HBV infection.

Three amino acid substitutions in the NS1 protein change the virus replication of H5N1 influenza virus in human cells.

Influenza A viruses have sophisticated strategies to promote their own replication. Here, we found that three H5N1 influenza viruses display different replication patterns in human A549 and macrophage cells. The HN01 virus displayed poor replication compared to HN021 and JS01. In addition, the HN01 virus was unable to counteract the interferon response and block general gene expression. This capability was restored by three amino acid substitutions on the NS1 protein: K55E, K66E, and C133F, resulting in recovered binding to CPSF30 and decreased interferon response activity. Furthermore, a recombinant HN01 virus expressing either NS1-C133F or the triple mutation replicate with higher titers in human A549 cells and macrophages compared to the parent virus. These three amino acid mutations reveal a new pathway to alter H5N1 virus replication.