A Guideline Strategy for Identifying a Viral Gene/Protein Evading Antiviral Innate Immunity.

Antiviral innate immunity is the first line of defence against viruses. The interferon (IFN) signaling pathway, the DNA damage response (DDR), apoptosis, endoplasmic reticulum (ER) stress, and autophagy are involved in antiviral innate immunity. Viruses abrogate the antiviral immune response of cells to replication in various ways. Viral genes/proteins play a key role in evading antiviral innate immunity. Here, we will discuss the interference of viruses with antiviral innate immunity and the strategy for identifying viral gene/protein immune evasion.

Intercellular Transport of Viral Proteins.

Viruses are vehicles to exchange genetic information and proteins between cells and organisms by infecting their target cells either cell-free, or depending on cell-cell contacts. Several viruses like certain retroviruses or herpesviruses transmit by both mechanisms. However, viruses have also evolved the properties to exchange proteins between cells independent of viral particle formation. This exchange of viral proteins can be directed to target cells prior to infection to interfere with restriction factors and intrinsic immunity, thus, making the target cell prone to infection. However, also bystander cells, e.g. immune cell populations, can be targeted by viral proteins to dampen antiviral responses. Mechanistically, viruses exploit several routes of cell-cell communication to exchange viral proteins like the formation of extracellular vesicles or the formation of long-distance connections like tunneling nanotubes. Although it is known that viral nucleic acids can be transferred between cells as well, this chapter concentrates on viral proteins of human pathogenic viruses covering all Baltimore classes and summarizes our current knowledge on intercellular transport of viral proteins between cells.

Nonstructural protein 4 of human norovirus self-assembles into various membrane-bridging multimers.

Single-stranded, positive-sense RNA ((+)RNA) viruses replicate their genomes in virus-induced intracellular membrane compartments. (+)RNA viruses dedicate a significant part of their small genomes (a few thousands to a few tens of thousands of bases) to the generation of these compartments by encoding membrane-interacting proteins and/or protein domains. Noroviruses are a very diverse genus of (+)RNA viruses including human and animal pathogens. Human noroviruses are the major cause of acute gastroenteritis worldwide, with genogroup II genotype 4 (GII.4) noroviruses accounting for the vast majority of infections. Three viral proteins encoded in the N-terminus of the viral replication polyprotein direct intracellular membrane rearrangements associated with norovirus replication. Of these three, nonstructural protein 4 (NS4) seems to be the most important, although its exact functions in replication organelle formation are unknown. Here we produce, purify and characterize GII.4 NS4. AlphaFold modeling combined with experimental data refine and correct our previous crude structural model of NS4. Using simple artificial liposomes, we report an extensive characterization of the membrane properties of NS4. We find that NS4 self-assembles and thereby bridges liposomes together. Cryo-EM, NMR and membrane flotation show formation of several distinct NS4 assemblies, at least two of them bridging pairs of membranes together in different fashions. Noroviruses belong to (+)RNA viruses whose replication compartment is extruded from the target endomembrane and generates double-membrane vesicles. Our data establish that the 21-kDa GII.4 human norovirus NS4 can, in the absence of any other factor, recapitulate in tubo several features, including membrane apposition, that occur in such processes.

The human retroviral-like aspartic protease 1 (ASPRV1): From in vitro studies to clinical correlations.

The human retroviral-like aspartic protease 1 (ASPRV1) is a retroviral-like protein that was first identified in the skin due to its expression in the stratum granulosum layer of the epidermis. Accordingly, it is also referred to as skin-specific aspartic protease. Similar to the retroviral polyproteins, the full-length ASPRV1 also undergoes self-proteolysis, the processing of the precursor is necessary for the autoactivation of the protease domain. ASPRV1's functions are well-established at the level of the skin: it is part of the epidermal proteolytic network and has a significant contribution to skin moisturization via the limited proteolysis of filaggrin; it is only natural protein substrate identified so far. Filaggrin and ASPRV1 are also specific for mammalians, these proteins provide unique features for the skins of these species, and the importance of filaggrin processing in hydration is proved by the fact that some ASPRV1 mutations are associated with skin diseases such as ichthyosis. ASPRV1 was also found to be expressed in macrophage-like neutrophil cells, indicating that its functions are not limited to the skin. In addition, differential expression of ASPRV1 was detected in many diseases, with yet unknown significance. The currently known enzymatic characteristics-that had been revealed mainly by in vitro studies-and correlations with pathogenic phenotypes imply potentially important functions in multiple cell types, which makes the protein a promising target of functional studies. In this review we describe the currently available knowledge and future perspective in regard to ASPRV1.

Temporal insights into molecular and cellular responses during rAAV production in HEK293T cells.

The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted an SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared with the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production.

Encephalomyocarditis Virus Structural Protein VP3 Interacts with MAVS and Promotes its Autophagic Degradation to Interfere with the Type I Interferon Signaling Pathway.

BACKGROUND: Understanding the mechanisms through which interferon (IFN) signaling is negatively regulated is crucial for preserving the equilibrium of innate immune reactions, as the innate immune system functions, such as the original barrier, combat threats to the host. Although the function of the encephalomyocarditis virus (EMCV) viral proteins in antagonizing innate immunity has been related to earlier studies, the precise mechanism underlying the role of viral protein 3 (VP3) in type I IFN has yet to be fully illuminated. METHODS: VP3 expression and many other adaptor molecules belonging to type I IFN pathway expression levels were evaluated using Western blotting. The IFN and other antiviral genes, such as interferon-stimulated genes (ISGs) 15 and 56, were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). A 50% tissue culture infectious dose (TCID50) assay was utilized to explore the effect of VP3 on EMCV proliferation in human embryonic kidney (HEK293) cells. Co-immunoprecipitation (Co-IP) assays and confocal microscope analysis were used to investigate the underlying mechanisms mediated by VP3. RESULTS: We discovered that the VP3 of EMCV acts as a suppressor of innate immune reactions. Increased levels of VP3 enhance viral reproduction through modulation of innate immune signaling pathways and suppression of antiviral responses. Additional information indicated that during viral infection, the VP3 of EMCV enhances autophagy and interacts specifically with mitochondrial antiviral signaling protein (MAVS), leading to its degradation in an autophagy pathway that relies on p62. CONCLUSIONS: Our findings showed that EMCV developed a tactic to combat host antiviral defenses by using autophagy to break down a protein that controls the innate immune response following a viral infection of the host. Notably, VP3 plays an important role in this process. Overall, these discoveries may provide a novel therapeutic target for EMCV.

Respiratory Syncytial Virus Vaccine Design Using Structure-Based Machine-Learning Models.

When designing live-attenuated respiratory syncytial virus (RSV) vaccine candidates, attenuating mutations can be developed through biologic selection or reverse-genetic manipulation and may include point mutations, codon and gene deletions, and genome rearrangements. Attenuation typically involves the reduction in virus replication, due to direct effects on viral structural and replicative machinery or viral factors that antagonize host defense or cause disease. However, attenuation must balance reduced replication and immunogenic antigen expression. In the present study, we explored a new approach in order to discover attenuating mutations. Specifically, we used protein structure modeling and computational methods to identify amino acid substitutions in the RSV nonstructural protein 1 (NS1) predicted to cause various levels of structural perturbation. Twelve different mutations predicted to alter the NS1 protein structure were introduced into infectious virus and analyzed in cell culture for effects on viral mRNA and protein expression, interferon and cytokine expression, and caspase activation. We found the use of structure-based machine learning to predict amino acid substitutions that reduce the thermodynamic stability of NS1 resulted in various levels of loss of NS1 function, exemplified by effects including reduced multi-cycle viral replication in cells competent for type I interferon, reduced expression of viral mRNAs and proteins, and increased interferon and apoptosis responses.

HIV1-Nef perturbs the integrity of blood testis barrier in rat model.

The blood-testis barrier is a specialized feature within the mammalian testis, located in close proximity to the basement membrane of seminiferous tubules. This barrier serves to divide the seminiferous epithelium into distinct basal and adluminal (apical) compartments. The selectivity of the BTB to foreign particles makes it a safe haven for the virus, and the high affinity of HIV for testis might lead to the vertical transmission of the virus. In the present study, recombinant HIV1-Nef (rNef) protein was injected intravenously to examine the effect of rNef on BTB. SD male rats received 250 µg and 500 µg of rNef along with 2% Evans blue dye within 1 ml through the tail vein. After 1 hour of perfusion, the animals were sacrificed for analysis. The dye migration assay and ELISA confirmed a significant impairment in the blood-testis barrier (BTB) and the manifestation of rNef in testes tissues, respectively. Moreover, a decline in the expression of tight junction proteins, including ZO1 and Occludin, was observed during rNef-induced BTB disruption. Overall, our findings demonstrated that rNef induces BTB disruption through various signaling events. At the site of ectoplasmic specialization of the seminiferous epithelium, the localization of cadherins was found to be disrupted, making the testis a vulnerable site. In conclusion, rNef perturbs the integrity of the blood-testis barrier in rat models; hence, it can also serve as a suitable model for studying the dynamics of the blood-testis barrier.

Established a rodent model to study the integrity of the blood testis barrier (BTB).Recombinant Nef (rNef) of HIV1 can breach the toughest physiological barrier of BTB.Integrity of BTB gets interrupted by rNef through the 'disengagement' and 'engagement' mechanisms of BTB dynamics.Major constituent proteins of BTB, including Occludin and ZO-1 were found to be highly disrupted by rNef; and seem to be the key aberrant for the compromised BTB.rNef also dislocated the localization of N & E cadherins in the rat testes; which would have affected the cadherin-based epithelial adhesion system of BTB and finally caused the breach.

Recent Advances in Lateral Flow Assays for Viral Protein Detection with Nanomaterial-Based Optical Sensors.

Controlling the progression of contagious diseases is crucial for public health management, emphasizing the importance of early viral infection diagnosis. In response, lateral flow assays (LFAs) have been successfully utilized in point-of-care (POC) testing, emerging as a viable alternative to more traditional diagnostic methods. Recent advancements in virus detection have primarily leveraged methods such as reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and the enzyme-linked immunosorbent assay (ELISA). Despite their proven effectiveness, these conventional techniques are often expensive, require specialized expertise, and consume a significant amount of time. In contrast, LFAs utilize nanomaterial-based optical sensing technologies, including colorimetric, fluorescence, and surface-enhanced Raman scattering (SERS), offering quick, straightforward analyses with minimal training and infrastructure requirements for detecting viral proteins in biological samples. This review describes the composition and mechanism of and recent advancements in LFAs for viral protein detection, categorizing them into colorimetric, fluorescent, and SERS-based techniques. Despite significant progress, developing a simple, stable, highly sensitive, and selective LFA system remains a formidable challenge. Nevertheless, an advanced LFA system promises not only to enhance clinical diagnostics but also to extend its utility to environmental monitoring and beyond, demonstrating its potential to revolutionize both healthcare and environmental safety.

Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly.

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3-48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

Updated Insights into the Phylogenetics, Phylodynamics, and Genetic Diversity of Nipah Virus (NiV).

Nipah virus (NiV), a biosafety level 4 agent, was first identified in human clinical cases during an outbreak in 1998 in Malaysia and Singapore. While flying foxes are the primary host and viral vector, the infection is associated with a severe clinical presentation in humans, resulting in a high mortality rate. Therefore, NiV is considered a virus with an elevated epidemic potential which is further underscored by its recent emergence (September 2023) as an outbreak in India. Given the situation, it is paramount to understand the molecular dynamics of the virus to shed more light on its evolution and prevent potential future outbreaks. In this study, we conducted Bayesian phylogenetic analysis on all available NiV complete genomes, including partial N-gene NiV sequences (≥1000 bp) in public databases since the first human case, registered in 1998. We observed the distribution of genomes into three main clades corresponding to the genotypes Malaysia, Bangladesh and India, with the Malaysian clade being the oldest in evolutionary terms. The Bayesian skyline plot showed a recent increase in the viral population size since 2019. Protein analysis showed the presence of specific protein families (Hendra_C) in bats that might keep the infection in an asymptomatic state in bats, which also serve as viral vectors. Our results further indicate a shortage of complete NiV genomes, which would be instrumental in gaining a better understanding of NiV's molecular evolution and preventing future outbreaks. Our investigation also underscores the critical need to strengthen genomic surveillance based on complete NiV genomes that will aid thorough genetic characterization of the circulating NiV strains and the phylogenetic relationships between the henipaviruses. This approach will better prepare us to tackle the challenges posed by the NiV virus and other emerging viruses.

Coronavirus takeover of host cell translation and intracellular antiviral response: a molecular perspective.

Coronaviruses are a group of related RNA viruses that cause respiratory diseases in humans and animals. Understanding the mechanisms of translation regulation during coronaviral infections is critical for developing antiviral therapies and preventing viral spread. Translation of the viral single-stranded RNA genome in the host cell cytoplasm is an essential step in the life cycle of coronaviruses, which affects the cellular mRNA translation landscape in many ways. Here we discuss various viral strategies of translation control, including how members of the Betacoronavirus genus shut down host cell translation and suppress host innate immune functions, as well as the role of the viral non-structural protein 1 (Nsp1) in the process. We also outline the fate of viral RNA, considering stress response mechanisms triggered in infected cells, and describe how unique viral RNA features contribute to programmed ribosomal -1 frameshifting, RNA editing, and translation shutdown evasion.

Enhancement of recombinant adeno-associated virus activity by improved stoichiometry and homogeneity of capsid protein assembly.

Studies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles. Capillary gel electrophoresis showed high-density particles possessing a designed DNA encapsidated in the capsid composed of (VP1 + VP2)/VP3 = 0.27, whereas low-density particles have the same DNA but with a different capsid composition of (VP1 + VP2)/VP3 = 0.31, supported by sedimentation velocity-analytical ultracentrifugation and charge detection-mass spectrometry. In vitro analysis demonstrated that the low-density particles had 8.9% higher transduction efficacy than that of the particles before fractionation. Further, based on our recent findings of VP3 clip, we created rAAV2 single amino acid variants of the transcription start methionine of VP3 (M203V) and VP3 clip (M211V). The rAAV2-M203V variant had homogeneous particles with higher (VP1+VP2)/VP3 values (0.35) and demonstrated 24.7% higher transduction efficacy compared with the wild type. This study successfully provided highly functional rAAV by the extensive fractionation from the mixture of rAAV2 full particles or by the single amino acid replacement.

Widespread amyloid aggregates formation by Zika virus proteins and peptides.

Viral pathogenesis typically involves numerous molecular mechanisms. Protein aggregation is a relatively unknown characteristic of viruses, despite the fact that viral proteins have been shown to form terminally misfolded forms. Zika virus (ZIKV) is a neurotropic one with the potential to cause neurodegeneration. Its protein amyloid aggregation may link the neurodegenerative component to the pathogenicity associated with the viral infection. Therefore, we investigated protein aggregation in the ZIKV proteome as a putative pathogenic route and one of the alternate pathways. We discovered that it contains numerous anticipated aggregation-prone regions in this investigation. To validate our prediction, we used a combination of supporting experimental techniques routinely used for morphological characterization and study of amyloid aggregates. Several ZIKV proteins and peptides, including the full-length envelope protein, its domain III (EDIII) and fusion peptide, Pr N-terminal peptide, NS1 ß-roll peptide, membrane-embedded signal peptide 2K, and cytosolic region of NS4B protein, were shown to be highly aggregating in our study. Because our findings show that viral proteins can form amyloids in vitro, we need to do a thorough functional study of these anticipated APRs to understand better the role of amyloids in the pathophysiology of ZIKV infection.

Inconspicuous Yet Indispensable: The Coronavirus Spike Transmembrane Domain.

Membrane-spanning portions of proteins' polypeptide chains are commonly known as their transmembrane domains (TMDs). The structural organisation and dynamic behaviour of TMDs from proteins of various families, be that receptors, ion channels, enzymes etc., have been under scrutiny on the part of the scientific community for the last few decades. The reason for such attention is that, apart from their obvious role as an "anchor" in ensuring the correct orientation of the protein's extra-membrane domains (in most cases functionally important), TMDs often actively and directly contribute to the operation of "the protein machine". They are capable of transmitting signals across the membrane, interacting with adjacent TMDs and membrane-proximal domains, as well as with various ligands, etc. Structural data on TMD arrangement are still fragmentary at best due to their complex molecular organisation as, most commonly, dynamic oligomers, as well as due to the challenges related to experimental studies thereof. Inter alia, this is especially true for viral fusion proteins, which have been the focus of numerous studies for quite some time, but have provoked unprecedented interest in view of the SARS-CoV-2 pandemic. However, despite numerous structure-centred studies of the spike (S) protein effectuating target cell entry in coronaviruses, structural data on the TMD as part of the entire spike protein are still incomplete, whereas this segment is known to be crucial to the spike's fusogenic activity. Therefore, in attempting to bring together currently available data on the structure and dynamics of spike proteins' TMDs, the present review aims to tackle a highly pertinent task and contribute to a better understanding of the molecular mechanisms underlying virus-mediated fusion, also offering a rationale for the design of novel efficacious methods for the treatment of infectious diseases caused by SARS-CoV-2 and related viruses.

Rotavirus capping enzyme VP3 inhibits interferon expression by inducing MAVS degradation during viral replication.

The binding of viral RNA to RIG-I-like receptors triggers the formation of mitochondrial antiviral signaling (MAVS) protein aggregates critical for interferon (IFN) expression. Several rotavirus strains have been shown to suppress IFN expression by inducing MAVS degradation. Relying on transient expression assays, previous studies reached different conclusions regarding the identity of the rotavirus protein responsible for MAVS degradation, suggesting it was an activity of the rotavirus capping enzyme VP3 or the interferon antagonist NSP1. Here, we have used recombinant SA11 rotaviruses to identify the endogenous viral protein responsible for MAVS degradation and to analyze how the attack on MAVS impacts IFN expression. The recombinant viruses included those expressing modified VP3 or NSP1 proteins deficient in the ability to induce the degradation of MAVS or interferon regulatory factor-3 (IRF3), or both. With these viruses, we determined that VP3 directs the proteasomal degradation of MAVS but plays no role in IRF3 degradation. Moreover, NSP1 was determined to induce IRF3 degradation but to have no impact on MAVS degradation. Analysis of rotavirus-infected cells indicated that IRF3 degradation was more efficient than MAVS degradation and that NSP1 was primarily responsible for suppressing IFN expression in infected cells. However, VP3-mediated MAVS degradation contributed to IFN suppression in cells that failed to produce functional NSP1, pointing to a subsidiary role for VP3 in the IFN antagonist activity of NSP1. Thus, VP3 is a multifunctional protein with several activities that counter anti-rotavirus innate immune responses, including capping of viral (+)RNAs, hydrolysis of the RNase L 2-5A (2'-5' oligoadenylate) signaling molecule, and proteasomal degradation of MAVS. IMPORTANCE Rotavirus is an enteric RNA virus that causes severe dehydrating gastroenteritis in infants and young children through infection of enterocytes in the small intestine. Timely clearance of the virus demands a robust innate immune response by cells associated with the small intestine, including the expression of interferon (IFN). Previous studies have shown that some rotavirus strains suppress the production of interferon, by inducing the degradation of mitochondrial antiviral signaling (MAVS) protein and interferon regulatory factor-3 (IRF3). In this study, we have used reverse genetics to generate recombinant rotaviruses expressing compromised forms of VP3 or NSP1, or both, to explore the function of these viral proteins in the degradation of MAVS and IRF3. Our results demonstrate that VP3 is responsible for MAVS depletion in rotavirus-infected cells, and through this activity, helps to suppress IFN production. Thus, VP3 functions to support the activity of rotavirus NSP1, the major interferon antagonist of the virus.

Synthesis and Screening of Chemical Agents Targeting Viral Protein Genome-Linked Protein of Telosma Mosaic Virus.

The viral protein genome-linked protein (VPg) of telosma mosaic virus (TeMV) plays an important role in viral reproduction. In this study, the expression conditions of TeMV VPg were explored. A series of novel benzenesulfonamide derivatives were synthesized. The binding sites of the target compounds and TeMV VPg were studied by molecular docking, and the interaction was verified by microscale thermophoresis. The study revealed that the optimal expression conditions for TeMV VPg were in Escherichia coli Rosetta with IPTG concentration of 0.8 mM and induction temperature of 25 °C. Compounds A4, A6, A9, A16, and A17 exhibited excellent binding affinity to TeMV VPg, with Kd values of 0.23, 0.034, 0.19, 0.086, and 0.22 µM, respectively. LYS 121 is the key amino acid site. Compounds A9 inhibited the expression of TeMV VPg in Nicotiana benthamiana. The results suggested that TeMV VPg is a potential antiviral target to screen anti-TeMV compounds.

Non-Structural Protein-W61 as a Novel Target in Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV): An In-Vitro and In-Silico Study on Protein-Protein Interactions with Nucleoprotein and Viral Replication.

The non-structural protein (NSs) and nucleoprotein (NP) of the severe fever with thrombocytopenia syndrome virus (SFTSV) encoded by the S segment are crucial for viral pathogenesis. They reside in viroplasm-like structures (VLS), but their interaction and their significance in viral propagation remain unclear. Here, we investigated the significance of the association between NSs and NP during viral infection through in-silico and in-vitro analyses. Through in-silico analysis, three possible binding sites were predicted, at positions C6S (Cystein at 6th position to Serine), W61Y (Tryptophan 61st to Tyrosine), and S207T (Serine 207th to Threonine), three mutants of NSs were developed by site-directed mutagenesis and tested for NP interaction by co-immunoprecipitation. NSsW61Y failed to interact with the nucleoprotein, which was substantiated by the conformational changes observed in the structural analyses. Additionally, molecular docking analysis corroborated that the NSW61Y mutant protein does not interact well compared to wild-type NSs. Over-expression of wild-type NSs in HeLa cells increased the SFTSV replication by five folds, but NSsW61Y exhibited 1.9-folds less viral replication than wild-type. We demonstrated that the W61Y alteration was implicated in the reduction of NSs-NP interaction and viral replication. Thus, the present study identified a critical NSs site, which could be targeted for development of therapeutic regimens against SFTSV.

Circulating foot-and-mouth disease virus serotype A African-genotype IV in Egypt during 2022.

Background and Aim: Foot-and-mouth disease (FMD) virus causes continuous outbreaks, leading to serious economic consequences that affect animal productivity and restrict trade movement. The potential influence of the disease was due to the emergence of new strains or re-emergence of local strains with major antigenic variations due to genetic mutations. This study aims to evaluate circulating virus in samples collected from infected animals during an outbreak using antigenic characterization and identify whether there is an emergence of a new strain or mutation. Materials and Methods: Reverse-transcription polymerase chain reaction (RT-PCR) was used to screen 86 samples. Viral protein 1 (VP1) codon sequencing was performed. The virus was isolated from the samples inoculated on the baby-hamster kidney cell line and Enzyme-linked immunosorbent assay was performed for serotyping and antigen detection. Results: Based on the RT-PCR screening results, 10 positive samples were selected for sequencing. The sequences belonged to the FMD serotype A African topotype originating from the ancestor prototype Sudan/77, with which it shared 98.48% ± 1.2% similarity. The divergence with local isolates from 2020 was 9.3%. In addition, the sequences were 96.84% ± 1.01% and 95.84% ± 0.79% related to Egyptian-Damietta type 2016 and Sudanese-2018, respectively. Divergence with vaccinal strains ranged from 10% to 17%. Amino acid sequence analysis revealed that the isolates had variation in the most prominent antigenic regions (residues 35-75) and the immunogenic determinants of the G-H loop of VP1 (residues 100-146 and 161-175). Conclusion: The current isolates should be included in the locally produced vaccine to provide broader immunogenic coverage against serotype A African topotypes.

Implications of AAV affinity column reuse and vector stability on product quality attributes.

In this work, the implications of AAV9 capsid design and column reuse on AAV9 vector product quality were assessed with POROS CaptureSelect (PCS) AAVX and AAV9 resins using sf9 insect cell-derived model AAV9 vectors with varying viral protein (VP) ratios. Chromatographic experiments with purified drug substance AAV9 model feeds indicated consistent vector elution profiles, independent of adeno-associated virus (AAV) VP ratio, or cycle number. In contrast, the presence of process impurities in the clarified lysate feeds resulted in clear changes in the elution patterns. This included increased aggregate content in the vector eluates over multiple cycles as well as clear differences in the performance of these affinity resin systems. The AAV9-serotype specific PCS AAV9 column, with lower vector elution pH, resulted in higher aggregate content over multiple cycles as compared to the serotype-independent PCS AAVX column. Further, the results with vectors of varying VP ratio indicated that while one vector type eluate displayed higher aggregation in both affinity columns over column reuse, the eluate with the other vector type did not exhibit changes in the aggregation profile. Interestingly, vector aggregates in the affinity eluates also contained double-stranded DNA impurities and histone proteins, with similar trends to the aggregate levels. This behavior upon column reuse indicates that these host cell impurities are likely carried over to subsequent runs due to incomplete clean-in-place (CIP). These results indicate that feed impurities, affinity resin characteristics, elution pH, column CIP, and vector stability can impact the reusability of AAV affinity columns and product quality.