This study proposes a novel approach to studying severe acute respiratory syndrome coronavirus 2 virus mutations through sequencing data comparison. Traditional consensus-based methods, which focus on the most common nucleotide at each position, might overlook or obscure the presence of low-frequency variants. Our method, in contrast, retains all sequenced nucleotides at each position, forming a genomic matrix. Utilizing simulated short reads from genomes with specified mutations, we contrasted our genomic matrix approach with the consensus sequence method. Our matrix methodology, across multiple simulated datasets, accurately reflected the known mutations with an average accuracy improvement of 20% over the consensus method. In real-world tests using data from GISAID and NCBI-SRA, our approach demonstrated an increase in reliability by reducing the error margin by approximately 15%. The genomic matrix approach offers a more accurate representation of the viral genomic diversity, thereby providing superior insights into virus evolution and epidemiology.

The HIV-1 Rev protein expressed in the early stage of virus replication is involved in the nuclear export of some forms of virus RNA. Naturally occurring polymorphisms in the Rev protein could influence its activity. The association between the genetic features of different virus variants and HIV infection pathogenesis has been discussed for many years. In this study, Rev diversity among HIV-1 group M clades was analyzed to note the signatures that could influence Rev activity and, subsequently, clinical characteristics. From the Los Alamos HIV Sequence Database, 4962 Rev sequences were downloaded and 26 clades in HIV-1 group M were analyzed for amino acid changes, conservation in consensus sequences, and the presence of clade-specific amino acid substitutions (CSSs) and the Wu-Kabat protein variability coefficient (WK). Subtypes G, CRF 02_AG, B, and A1 showed the largest amino acid changes and diversity. The mean conservation of the Rev protein was 80.8%. In consensus sequences, signatures that could influence Rev activity were detected. In 15 out of 26 consensus sequences, an insertion associated with the reduced export activity of the Rev protein, 95QSQGTET96, was identified. A total of 32 CSSs were found in 16 clades, wherein A6 had the 41Q substitution in the functionally significant region of Rev. The high values of WK coefficient in sites 51 and 82, located on the Rev interaction surface, indicate the susceptibility of these positions to evolutionary replacements. Thus, the noted signatures require further investigation.

A protein sequence encodes its energy landscape-all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs-questioning the differences between them and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the consensus protein derived from our full Ribonuclease H sequence alignment is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted set of sequences is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order sequence correlations using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

An effective HIV-1 vaccine must elicit broadly neutralizing antibodies (bnAbs) against highly diverse Envelope glycoproteins (Env). Since Env with the longest hypervariable (HV) loops is more resistant to the cognate bnAbs than Env with shorter HV loops, we redesigned hypervariable loops for updated Env consensus sequences of subtypes B and C and CRF01_AE. Using modeling with AlphaFold2, we reduced the length of V1, V2, and V5 HV loops while maintaining the integrity of the Env structure and glycan shield, and modified the V4 HV loop. Spacers are designed to limit strain-specific targeting. All updated Env are infectious as pseudoviruses. Preliminary structural characterization suggests that the modified HV loops have a limited impact on Env\'s conformation. Binding assays show improved binding to modified subtype B and CRF01_AE Env but not to subtype C Env. Neutralization assays show increases in sensitivity to bnAbs, although not always consistently across clades. Strikingly, the HV loop modification renders the resistant CRF01_AE Env sensitive to 10-1074 despite the absence of a glycan at N332.

High-mannose-type glycan structure of N-glycoproteins plays important roles in the proper folding of proteins in sorting glycoprotein secretion and degradation of misfolded proteins in the endoplasmic reticulum (ER). The Glc1Man9GlcNAc2 (G1M9)-type N-glycan is one of the most important signaling molecules in the ER. However, current chemical synthesis strategies are laborious, warranting more practical approaches for G1M9-glycopeptide development. Wang et al. reported the procedure to give G1M9-Asn-Fmoc through chemical modifications and purifications from 40 chicken eggs, but only 3.3 mg of G1M9-glycopeptide was obtained. Therefore, better methods are needed to obtain more than 10 mg of G1M9-glycopeptide. In this study, we report the preparation of G1M9-glycopeptide (13.2 mg) linking Asn-Gly-Thr triad as consensus sequence from 40 chicken eggs. In this procedure, λ-carrageenan treatment followed by papain treatment was used to separate the Fc region of IgY antibody that harbors high-mannose glycans. Moreover, cotton hydrophilic interaction liquid chromatography was adapted for easy purification. The resulting G1M9-Asn(Fmoc)-Gly-Thr was identified by nuclear magnetic resonance and mass spectroscopy. G1M9-Asn(Fmoc)-Gly, G1M9-Asn(Fmoc), and G1M9-OH were also detected by mass spectroscopy. Here, our developed G1M9-tripeptide might be useful for the elucidation of glycoprotein functions as well as the specific roles of the consensus sequence.

OBJECTIVE: This study aimed to identify by in silico methods tropomyosin consensus B and T epitopes of shrimp species, house dust mites, insects, and nematodes associated with allergic diseases in tropical countries.
METHODS: In silico analysis included tropomyosin from mites (Der p 10, Der f 10, Blo t 10), insects (Aed a 10, Per a 7, Bla g 7), shrimp (Lit v 1, Pen m 1, Pen a 1), and nematode (Asc l 3) all sequences were taken from the UniProt database. Linear IgE epitopes were predicted with AlgPred 2.0 and validated with BepiPred 3.0. MHC-II binding T cell epitopes were predicted using the IEDB server, which implements nine predictive methods (consensus method, combinatorial library, NN-align-2.3, NN- align-2.2, SMM-align, Sturniolo, NetMHCIIpan 3.1, and NetMHCIIpan 3.2) these predictions focused on 10 HLA-DR and 2 HLA-DQ alleles associated with allergic diseases. Subsequently, consensus B and T epitopes present in all species were identified.
RESULTS: We identified 12 sequences that behaved as IgE-epitopes and B-cell epitopes, three of them: 160RKYDEVARKLAMVEA174, 192ELEEELRVVGNNLKSLEVSEEKAN215, 251KEVDRLEDELV261 were consensus in all species. Eleven peptides (T-epitopes) showed strong binding (percentile rank ≤ 2.0) to HLA-DRB1*0301, *0402, *0411, *0701, *1101, *1401, HLA-DQA1*03:01/DQB1*03:02, and HLA- DQA1*05:01/DQB1*02:01. Only two T-epitopes were consensus in all species: 167RKLAMVEADLERAEERAEt GEsKIVELEEELRV199, and 218EEeY KQQIKT LTaKLKEAEARAEFAERSV246. Subsequently, we identified 2 B and T epitope sequences and reached a consensus between species 167RKLAMVEA174 and 192ELEEELRV199.
CONCLUSIONS: These data describe three sequences that may explain the IgE cross-reactivity between the analyzed species. In addition, the consensus B and T epitopes can be used for further in vitro investigations and may help to design multiple-epitope protein-based immunotherapy for tropomyosin-related allergic diseases.
OBJECTIVE: Este estudio tuvo como objetivo identificar mediante métodos in silico epítopes B y T consenso de tropomiosina de especies de camarón, ácaros del polvo doméstico, insectos y nematodos asociados a enfermedades alérgicas en países tropicales.
UNASSIGNED: El análisis in silico incluyó tropomiosina de ácaros (Der p 10, Der f 10, Blo t 10), insectos (Aed a 10, Per a 7, Bla g 7), camarones (Lit v 1, Pen m 1, Pen a 1), y nematodo (Asc l 3). Todas las secuencias se tomaron de la base de datos UniProt. Los epítopes IgE lineales se predijeron con AlgPred 2.0 y se validaron con BepiPred 3.0. Los epítopes de células T de unión a MHC-II se predijeron utilizando el servidor IEDB, que implementa nueve métodos predictivos (método de consenso, biblioteca combinatoria, NN-align-2.3, NN-align-2.2, SMM-align, Sturniolo, NetMHCIIpan 3.1 y NetMHCIIpan 3.2). Estas predicciones se centraron en diez alelos HLA-DR y 2 HLA-DQ asociados con enfermedades alérgicas. Posteriormente, se identificaron epítopes consenso B y T presentes en todas las especies.
RESULTS: Se identificaron 12 secuencias que se comportaron como epítopes de IgE y, también, como epítopes de células B. Tres de ellas: 160RKYDEVARKLAMVEA174, 192ELEEELRVVGNNLKSLEVSEEKAN213 y 251KEVDRLEDELV261, fueron consenso en todas las especies. Once péptidos mostraron una fuerte unión (rango percentil ≤ 2,0) a HLA-DRB1*0301, *0402, *0411, *0701, *1101, *1401 y a HLA HLA-DQA1*03:01/DQB1*03:02, o HLA-DQA1*05:01/DQB1*02:01. Solo se encontraron dos secuencias: 167RKLAMVEADLERAEERAEtGEsKIVELEEELRV199 con fuerte afinidad por HLA-DQA1*03:01/DQB1*03:02, y HLA-DQA1*05:01/DQB1*02:01. Se identificaron dos secuencias que son epítopos B y T, y son consenso entre especies: 167RKLAMVEA174 y 192ELEEELRV199.
CONCLUSIONS: Estos datos describen tres secuencias que pueden explicar la reactividad cruzada de IgE entre las especies analizadas. Además, los epítopos B y T consenso se pueden usar para investigaciones in vitro adicionales, y pueden ayudar a diseñar inmunoterapia basada en proteínas de múltiepítopes para enfermedades alérgicas relacionadas con la tropomiosina.

Nearly 300 million individuals live with chronic hepatitis B virus (HBV) infection (CHB), for which no curative therapy is available. As viral diversity is associated with pathogenesis and immunological control of infection, improved methods to characterize this diversity could aid drug development efforts. Conventionally, viral sequencing data are mapped/aligned to a reference genome, and only the aligned sequences are retained for analysis. Thus, reference selection is critical, yet selecting the most representative reference a priori remains difficult. We investigate an alternative pangenome approach which can combine multiple reference sequences into a graph which can be used during alignment. Using simulated short-read sequencing data generated from publicly available HBV genomes and real sequencing data from an individual living with CHB, we demonstrate alignment to a phylogenetically representative \'genome graph\' can improve alignment, avoid issues of reference ambiguity, and facilitate the construction of sample-specific consensus sequences more genetically similar to the individual\'s infection. Graph-based methods can, therefore, improve efforts to characterize the genetics of viral pathogens, including HBV, and have broader implications in host-pathogen research.

In this study, the phenomenon of the stability-activity trade-off, which is increasingly recognized in enzyme engineering, was explored. Typically, enhanced stability in enzymes correlates with diminished activity. Utilizing Rosa roxburghii copper-zinc superoxide dismutase (RrCuZnSOD) as a model, single-site mutations were introduced based on a semirational design derived from consensus sequences. The initial set of mutants was selected based on activity, followed by combinatorial mutation. This approach yielded two double-site mutants, D25/A115T (18,688 ± 206 U/mg) and A115T/S135P (18,095 ± 1556 U/mg), exhibiting superior enzymatic properties due to additive and synergistic effects. These mutants demonstrated increased half-lives (T1/2) at 80 °C by 1.2- and 1.6-fold, respectively, and their melting temperatures (Tm) rose by 3.4 and 2.5 °C, respectively, without any loss in activity relative to the wild type. Via an integration of structural analysis and molecular dynamics simulations, we elucidated the underlying mechanism facilitating the concurrent enhancement of both thermostability and enzymatic activity.

The prevalence of porcine reproductive and respiratory syndrome virus 1 (PRRSV1) isolates has continued to increase in Chinese swine herds in recent years. However, no effective control strategy is available for PRRSV1 infection in China. In this study, we generated the first infectious cDNA clone (rHLJB1) of a Chinese PRRSV1 isolate and subsequently used it as a backbone to construct an ORF2-6 chimeric virus (ORF2-6-CON). This virus contained a synthesized consensus sequence of the PRRSV1 ORF2-6 gene encoding all the envelope proteins. The ORF2-6 consensus sequence shared > 90% nucleotide similarity with four representative strains (Amervac, BJEU06-1, HKEU16 and NMEU09-1) of PRRSV1 in China. ORF2-6-CON had replication efficacy similar to that of the backbone rHLJB1 virus in primary alveolar macrophages (PAMs) and exhibited cell tropism in Marc-145 cells. Piglet inoculation and challenge studies indicated that ORF2-6-CON is not pathogenic to piglets and can induce enhanced cross-protection against a heterologous SD1291 isolate. Notably, ORF2-6-CON inoculation induced higher levels of heterologous neutralizing antibodies (nAbs) against SD1291 than rHLJB1 inoculation, which was concurrent with a higher percentage of T follicular helper (Tfh) cells in tracheobronchial lymph nodes (TBLNs), providing the first clue that porcine Tfh cells are correlated with heterologous PRRSV nAb responses. The number of SD1291-strain-specific IFNγ-secreting cells was similar in ORF2-6-CON-inoculated and rHLJB1-inoculated pigs. Overall, our findings support that the Marc-145-adapted ORF2-6-CON can trigger Tfh cell and heterologous nAb responses to confer improved cross-protection and may serve as a candidate strain for the development of a cross-protective PRRSV1 vaccine.

The extended cleavage specificities of two hematopoietic serine proteases originating from the ray-finned fish, the spotted gar (Lepisosteus oculatus), have been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated using a panel of recombinant substrates. For one of the enzymes, the gar granzyme G, a strict preference for the aromatic amino acid Tyr was observed at the cleavable P1 position. Using a set of recombinant substrates showed that the gar granzyme G had a high selectivity for Tyr but a lower activity for cleaving after Phe but not after Trp. Instead, the second enzyme, gar DDN1, showed a high preference for Leu in the P1 position of substrates. This latter enzyme also showed a high preference for Pro in the P2 position and Arg in both P4 and P5 positions. The selectivity for the two Arg residues in positions P4 and P5 suggests a highly specific substrate selectivity of this enzyme. The screening of the gar proteome with the consensus sequences obtained by substrate phage display for these two proteases resulted in a very diverse set of potential targets. Due to this diversity, a clear candidate for a specific immune function of these two enzymes cannot yet be identified. Antisera developed against the recombinant gar enzymes were used to study their tissue distribution. Tissue sections from juvenile fish showed the expression of both proteases in cells in Peyer\'s patch-like structures in the intestinal region, indicating they may be expressed in T or NK cells. However, due to the lack of antibodies to specific surface markers in the gar, it has not been possible to specify the exact cellular origin. A marked difference in abundance was observed for the two proteases where gar DDN1 was expressed at higher levels than gar granzyme G. However, both appear to be expressed in the same or similar cells, having a lymphocyte-like appearance.