Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution.

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.

The Ebola virus VP40 matrix layer undergoes endosomal disassembly essential for membrane fusion.

Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

The RBPome of influenza A virus NP-mRNA reveals a role for TDP-43 in viral replication.

Genome-wide approaches have significantly advanced our knowledge of the repertoire of RNA-binding proteins (RBPs) that associate with cellular polyadenylated mRNAs within eukaryotic cells. Recent studies focusing on the RBP interactomes of viral mRNAs, notably SARS-Cov-2, have revealed both similarities and differences between the RBP profiles of viral and cellular mRNAs. However, the RBPome of influenza virus mRNAs remains unexplored. Herein, we identify RBPs that associate with the viral mRNA encoding the nucleoprotein (NP) of an influenza A virus. Focusing on TDP-43, we show that it binds several influenza mRNAs beyond the NP-mRNA, and that its depletion results in lower levels of viral mRNAs and proteins within infected cells, and a decreased yield of infectious viral particles. We provide evidence that the viral polymerase recruits TDP-43 onto viral mRNAs through a direct interaction with the disordered C-terminal domain of TDP-43. Notably, other RBPs found to be associated with influenza virus mRNAs also interact with the viral polymerase, which points to a role of the polymerase in orchestrating the assembly of viral messenger ribonucleoproteins.

Minor electrostatic changes robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry.

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

N6-methyladenosine modification of the 5' epsilon structure of the HBV pregenome RNA regulates its encapsidation by the viral core protein.

Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5' end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m6A)-modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m6A modification of the 5' ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m6A site mutant in the HBV 5' epsilon, we present evidence that m6A methylation of 5' epsilon is necessary for its encapsidation. The m6A modification of 5' epsilon increased the efficiency of viral RNA packaging, whereas the m6A of 3' epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m6A modification at 5' epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5' epsilon m6A site-mutated pgRNA failed to interact. HBV polymerase interaction with 5' epsilon was independent of m6A modification of 5' epsilon. This study highlights yet another pivotal role of m6A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA-protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m6A modification.

Hepatitis C virus core protein uses triacylglycerols to fold onto the endoplasmic reticulum membrane.

Lipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of core, which subsequently relocalizes to contiguous ER regions. During this motion, core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer.

Lipid environment modulates processivity and kinetics of a presenilin homolog acting on multiple substrates in vitro.

Intramembrane proteases (IPs) hydrolyze peptides in the lipid membrane. IPs participate in a number of cellular pathways including immune response and surveillance, and cholesterol biosynthesis, and they are exploited by viruses for replication. Despite their broad importance across biology, how activity is regulated in the cell to control protein maturation and release of specific bioactive peptides at the right place and right time remains largely unanswered, particularly for the intramembrane aspartyl protease (IAP) subtype. At a molecular biochemical level, different IAP homologs can cleave non-biological substrates, and there is no sequence recognition motif among the nearly 150 substrates identified for just one IAP, presenilin-1, the catalytic component of γ-secretase known for its involvement in the production of amyloid-ß plaques associated with Alzheimer disease. Here we used gel-based assays combined with quantitative mass spectrometry and FRET-based kinetics assays to probe the cleavage profile of the presenilin homolog from the methanogen Methanoculleus marisnigri JR1 as a function of the surrounding lipid-mimicking environment, either detergent micelles or bicelles. We selected four biological IAP substrates that have not undergone extensive cleavage profiling previously, namely, the viral core protein of Hepatitis C virus, the viral core protein of Classical Swine Fever virus, the transmembrane segment of Notch-1, and the tyrosine receptor kinase ErbB4. Our study demonstrates a proclivity toward cleavage of substrates at positions of low average hydrophobicity and a consistent role for the lipid environment in modulating kinetic properties.

G-quadruplex in hepatitis B virus pregenomic RNA promotes its translation.

Hepatitis B virus (HBV) is a hepatotropic DNA virus that has a very compact genome. Due to this genomic density, several distinct mechanisms are used to facilitate the viral life cycle. Recently, accumulating evidence show that G-quadruplex (G4) in different viruses play essential regulatory roles in key steps of the viral life cycle. Although G4 structures in the HBV genome have been reported, their function in HBV replication remains elusive. In this study, we treated an HBV replication-competent cell line and HBV-infected cells with the G4 structure stabilizer pyridostatin (PDS) and evaluated different HBV replication markers to better understand the role played by the G4. In both models, we found PDS had no effect on viral precore RNA (pcRNA) or pre-genomic RNA (pgRNA), but treatment did increase HBeAg/HBc ELISA reads and intracellular levels of viral core/capsid protein (HBc) in a dose-dependent manner, suggesting post-transcriptional regulation. To further dissect the mechanism of G4 involvement, we used in vitro-synthesized HBV pcRNA and pgRNA. Interestingly, we found PDS treatment only enhanced HBc expression from pgRNA but not HBeAg expression from pcRNA. Our bioinformatic analysis and CD spectroscopy revealed that pgRNA harbors a conserved G4 structure. Finally, we introduced point mutations in pgRNA to disrupt its G4 structure and observed the resulting mutant failed to respond to PDS treatment and decreased HBc level in in vitro translation assay. Taken together, our data demonstrate that HBV pgRNA contains a G4 structure that plays a vital role in the regulation of viral mRNA translation.

Selective depletion of HBV-infected hepatocytes by class A capsid assembly modulators requires high levels of intrahepatic HBV core protein.

Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.

Rotavirus core shell protein sites that regulate intra-particle polymerase activity.

IMPORTANCE: Rotaviruses are important causes of severe gastroenteritis in young children. A characteristic feature of rotaviruses is that they copy ribonucleic acid (RNA) inside of the viral particle. In fact, the viral polymerase (VP1) only functions when it is connected to the viral inner core shell protein (VP2). Here, we employed a biochemical assay to identify which sites of VP2 are critical for regulating VP1 activity. Specifically, we engineered VP2 proteins to contain amino acid changes at structurally defined sites and assayed them for their capacity to support VP1 function in a test tube. Through this work, we were able to identify several VP2 residues that appeared to regulate the activity of the polymerase, positively and negatively. These results are important because they help explain how rotavirus synthesizes its RNA while inside of particles and they identify targets for the future rational design of drugs to prevent rotavirus disease.

Single-molecule-binding studies of antivirals targeting the hepatitis C virus core protein.

IMPORTANCE: The hepatitis C virus is associated with nearly 300,000 deaths annually. At the core of the virus is an RNA-protein complex called the nucleocapsid, which consists of the viral genome and many copies of the core protein. Because the assembly of the nucleocapsid is a critical step in viral replication, a considerable amount of effort has been devoted to identifying antiviral therapeutics that can bind to the core protein and disrupt assembly. Although several candidates have been identified, little is known about how they interact with the core protein or how those interactions alter the structure and thus the function of this viral protein. Our work biochemically characterizes several of these binding interactions, highlighting both similarities and differences as well as strengths and weaknesses. These insights bolster the notion that this viral protein is a viable target for novel therapeutics and will help to guide future developments of these candidate antivirals.

Avian Reovirus σA Protein Inhibits Type I Interferon Production by Abrogating Interferon Regulatory Factor 7 Activation.

Type I interferon (IFN) response is the first line of host-based innate immune defense against viral infections. However, viruses have developed multiple strategies to counter host IFN responses, so they may continue infecting hosts via effective replication. Avian reovirus (ARV), an RNA virus, causes viral arthritis or tenosynovitis in chickens. Previous studies have shown that ARV is highly resistant to the antiviral effects of IFN. However, the underlying mechanisms that enable ARV to block the IFN pathway remain unclear. In this study, we found that ectopic expression of ARV protein, σA, significantly inhibited the production of IFN-ß induced by melanoma-differentiation-associated gene 5 (MDA5) and poly(I·C). Knockdown of σA during ARV infection enhances the IFN-ß response and suppresses viral replication. ARV σA inhibited the MDA5-mediated IFN-ß activation by targeting interferon regulatory factor 7 (IRF7). Further studies demonstrated that σA interacts with IRF7, thereby blocking IRF7 dimerization and nuclear translocation, finally leading to the inhibition of IFN-ß production. These findings reveal a novel mechanism that allows ARV to evade host antiviral immunity. IMPORTANCE ARV, the causative agent of viral arthritis or tenosynovitis in chickens, has a significant economic impact as it results in poor weight gain and increased feed conversion ratios. The MDA5-mediated IFN-ß signal pathway plays an important role in host antiviral defense. Therefore, RNA viruses have developed mechanisms to counter this signaling pathway and successfully establish infection. However, the strategies adopted by ARV to block MDA5-IRF7 signaling remain unclear. In the current study, we demonstrated that ARV σA inhibits this pathway by binding to IRF7, which blocked IRF7 dimerization and nuclear translocation. Our findings may provide insights into how avian reovirus counteracts the innate antiviral immunity of the host to ensure viral replication.

Amino acid substitutions in norovirus VP1 dictate host dissemination via variations in cellular attachment.

IMPORTANCE: All viruses initiate infection by utilizing receptors to attach to target host cells. These virus-receptor interactions can therefore dictate viral replication and pathogenesis. Understanding the nature of virus-receptor interactions could also be important for the development of novel therapies. Noroviruses are non-enveloped icosahedral viruses of medical importance. They are a common cause of acute gastroenteritis with no approved vaccine or therapy and are a tractable model for studying fundamental virus biology. In this study, we utilized the murine norovirus model system to show that variation in a single amino acid of the major capsid protein alone can affect viral infectivity through improved attachment to suspension cells. Modulating plasma membrane mobility reduced infectivity, suggesting an importance of membrane mobility for receptor recruitment and/or receptor conformation. Furthermore, different substitutions at this site altered viral tissue distribution in a murine model, illustrating how in-host capsid evolution could influence viral infectivity and/or immune evasion.

N-terminal domain of classical swine fever virus Npro induces proteasomal degradation of specificity protein 1 with reduced HDAC1 expression to evade from innate immune responses.

IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.

Performance of HCV core antigen and PCR testing in a predominantly genotype 3 population.

Hepatitis C core antigen (HCVcAg) is becoming increasingly recognized as an alternative to molecular testing for the confirmation of chronic hepatitis C. However, there are limited data on the performance of this assay in a genotype 3 (GT3) predominant country like Pakistan. We conducted a study to evaluate the diagnostic performance of HCVcAg against the HCV polymerase chain reaction (PCR) molecular test. HCV antibody-positive patients requiring confirmatory testing were recruited from August to October 2018 at the Pakistan Kidney and Liver Institute and Research Center (PKLI&RC), Lahore, Pakistan. Patients with previously known diagnoses or treatment histories were excluded. The Abbott HCV Ag assay was used for HCVcAg testing. Results ≥3.00 fmol/L were considered positive for HCVcAg. The Abbott RealTime HCV assay was used for PCR testing with a lower detection limit of ≥12 IU/mL. We computed the sensitivity, specificity and correlation of HCVcAg against HCV PCR. A total of 394 patients were recruited. The median age of the patients was 42 years. Most participants were females (51.5%, n = 203), 30.7% (n = 121) had HTN, 10.4% DM (n = 41) and 5% had APRI ≥2. The overall sensitivity was 98.0% and the specificity was 98.6%. The lowest detection limit of cAg was an HCV RNA value of 4657 IU/mL. The levels of cAg were highly correlated with those of HCV RNA by Spearman's rank correlation test (r = 0.935, p < .001). HCVcAg represents a suitable alternative with high sensitivity and specificity compared with HCV PCR in the GT3-predominant population and can be incorporated into algorithms to improve linkage to care.

Synthesis of the full-length hepatitis B virus core protein and its capsid formation.

Chronic infection with hepatitis B virus (HBV) is a major cause of cirrhosis and liver cancer. Capsid assembly modulators can induce error-prone assembly of HBV core proteins to prevent the formation of infectious virions, representing promising candidates for treating chronic HBV infections. To explore novel capsid assembly modulators from unexplored mirror-image libraries of natural products, we have investigated the synthetic process of the HBV core protein for preparing the mirror-image target protein. In this report, the chemical synthesis of full-length HBV core protein (Cp183) containing an arginine-rich nucleic acid-binding domain at the C-terminus is presented. Sequential ligations using four peptide segments enabled the synthesis of Cp183 via convergent and C-to-N direction approaches. After refolding under appropriate conditions, followed by the addition of nucleic acid, the synthetic Cp183 assembled into capsid-like particles.

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope.

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15-gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

Hepatitis C virus modulates signal peptide peptidase to alter host protein processing.

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8+ T cells. The expression of MHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses.

Two Phylogenetic Cohorts of the Nucleocapsid Protein NP and Their Correlation with the Host Range of Influenza A Viruses.

Influenza A virus has a wide natural areal among birds, mammals, and humans. One of the main regulatory adaptors of the virus host range is the major NP protein of the viral nucleocapsid. Phylogenetic analysis of the NP protein of different viruses has revealed the existence of two phylogenetic cohorts in human influenza virus population. Cohort I includes classical human viruses that caused epidemics in 1957, 1968, 1977. Cohort II includes the H1N1/2009pdm virus, which had a mixed avian-swine origin but caused global human pandemic. Also, the highly virulent H5N1 avian influenza virus emerged in 2021 and caused outbreaks of lethal infections in mammals including humans, appeared to have the NP gene of the second phylogenetic cohort and, therefore, by the type of adaptation to human is similar to the H1N1/2009pdm virus and seems to possess a high epidemic potential for humans. The data obtained shed light on pathways and dynamics of adaptation of avian influenza viruses to humans and propose phylogenetic algorithm for systemic monitoring of dangerous virus strains to predict epidemic harbingers and take immediate preventive measures.