Abstract: Background: Persistent high-risk human papillomavirus (hrHPV) infection causes over 99% of cervical precancers and cancers worldwide, with HPV genotype 16 (HPV16) responsible for 50% of the cases. Latvia ranks among the top EU countries for cervical cancer incidence and mortality. In the general Latvian population, 4.2% women are hrHPV-infected, mostly with HPV16. However, information on the circulating HPV16 isolates is missing. Objectives: To study the genomic variability of the Latvian HPV16 isolates, compare them with HPV16 in Europe and across the globe, reveal features associated with the severity of cervical disease and uncover eventual sequence changes due the national HPV vaccination. Methods: DNA was extracted from the formalin-fixed paraffin-embedded cervical tissues of women diagnosed with cervical intraepithelial neoplasia (CIN) stages I-III and squamous cell carcinoma (SCC) grades 1-3 collected between 2012 and 2024. Samples positive for HPV16 were subjected to whole genome sequencing (WGS) on the Illumina platform (n=16) or Sanger sequencing of the E6/E7 coding region (n=31). Consensus HPV16 sequence was generated, and single nucleotide polymorphisms (SNP) and eventual amino acid substitutions (AAS) were analysed. Results: Complete genomes of 16 HPV16 variants were reconstructed, 13 related to the European sublineage A1, and 3, to the sublineage A2 references. Sequences showed high conservation, still 93 non-redundant variants were identified. The highest variability was observed for the capsid protein L2, the lowest, for oncoprotein E7. Prevalence of SNPs and AAS in the Latvian HPV16 variants, specifically in capsid protein L1, did not increase with time, showing no effect of HPV vaccination. Associations between HPV16 sequence features and severity of cervical disease were limited to AAS E6:L90V which was significantly more common in SCC grade 2/3 than in CINII/III cases (p=0.015). Conclusions: Highly conserved HPV16 genomes circulating in Latvia harbour series of unique, as well as common nonsynonymous SNPs with respective AAS, with one, AAS E6:L90V, associating with disease severity. No HPV vaccine escape variants were detected. Deciphering complete genomes of HPV16 from CIN and SCC cases in Latvia informs public authorities performing HPV vaccination and is useful for management of HPV-associated cervical diseases.

Abstract: Background/Objectives: Parvovirus infection cause sever diseases in both feline and ca-nine species, mostly affects adult cats and dogs, but cause higher risk in the kittens and puppies. This virus is known to be contagious; the simple way of spreading occurs through food and shelter as well as hands and clothing of people. The recovered species may continue to shed parvovirus in their feces for an extended period, leading to severe environmental contamination. There is no universal vaccine available that protect dogs and cats against parvovirus infections. The main goal of this study is to design a pan parvovirus multiepitope DNA based vaccine that could be administered to dog and cats. Methods: We utilized AI-machine learning incorporated server tools such as IEDB and NetMHCpan to predict B-cell and T-cell epitopes. VaxiJen and ToxinPred were used to analyze immune characteristic features and docking with feline alleles using HADdock server. Following, the immune response and stability of vaccine construct was confirmed with disulfide engineering, Normal mode analysis and molecular docking, and dynamic simulations were done with Toll like receptors of both feline and canine (TLR4 and TLR5) for 50ns. The triggered immune response was determined with immuno-simulation (ImmSim) and their activity in biological environment was reinforced with in silico cloning. Results: The B cell epitopes (NS1 - 9, NS2 - 4, VP1- 12 and VP2- 9) predicted with IEDB database were subjected to antigenicity prediction. MHC class I and IFN prediction and MHC class II and IL-4 prediction were done with IEDB and NetMHCpan. The T cell epitopes showed high binding affinities with the feline alleles. The final vaccine was de-signed by combining the top-ranked B-cell epitopes T- cell epitopes, filtered from high antigenic, non-allergic, non-toxic and good solubility, and with the better binding affinity score of the structural and non- structural proteins (NS1, NS2, VP1 and VP2) of feline and canine parvoviruses through linkers and adjuvants. The disulfide bond prediction and Normal mode analysis showed our vaccine construct are stable and flexible. The molecular docking analysis was performed between the designed vaccine epitopes and the TLRs (TLR4 – feline and TLR5 – canine) with Biovia Discovery Studio using Zdocker, it showed the better binding interaction with value of 22.26 (Zdock score), -47.409 (Zrank score) for feline and 16.54 (Zdock score), -134.295 (Zrank score) for canine. Conclusions: The pan multi-epitope DNA based vaccine combining the four major structural proteins (NS1, NS2, VP1 and VP2) possess dual purpose to protect both feline and canine species against the parvovirus were designed and constructed. The molecular docking and dynamic simulation analysis showed higher binding affinities and stable conformation with canine (TLR5) and feline (TLR4) toll like receptors. Though computational analysis will support us to predict the more precise top-ranked epitopes and their immuno-antigenic properties, further experimental validation will be required to be used against those viruses.

Abstract: Bacteriophages are viruses that attack bacterial cells, resulting in their lysis. Since the emergence of antibiotic resistance, phages have gained attention from the scientific community and are being explored for their potential use as anti-microbial agents. Since their discovery, naturally occurring wild-type phages have been used as anti-microbial agents. Though usage of antibiotics is confronted with problems, antibiotic resistance being the most pronounced, phages also have limitations, such as harboring toxin genes, not always lytic in nature, being involved in transduction, not having a broad host range, having a short life in the body due to mammalian immune response, and being attacked by bacterial anti-phage defense mechanisms. Modern approaches to phage therapy can address these problems associated with conventional phage therapy. These modern approaches include genetic phage engineering and using phage-derived enzymes, such as depolymerases and endolysins, in their wild-type form as well as in genetically engineered form. Genetic engineering has been performed on lytic, lysogenic, and filamentous phages for desired targeted results. In this review, we discuss the previous studies that have investigated these modern approaches for phage therapy against bacterial infections. Advantages and disadvantages of phage therapy are being discussed, and it is discussed how the use of modern approaches can affect different aspects of phage therapy.

Abstract: Vanilla planifolia, a high-value tropical orchid, is significantly impacted by viral pathogens that threaten its cultivation and productivity. This study employs metagenomic technique to detect and characterize the viral communities associated with V. planifolia in south Florida. Using high-throughput RNA sequencing, the Cymbidium mosaic virus (CymMV) and Vanilla latent virus (VLV) were prevalent in the plant system, with CymMV being the dominant viral species. Phylogenetic analysis of the CymMV coat protein gene revealed notable genetic divergence in the Homestead isolate, forming a distinct clade from global reference strains, suggesting local adaptation or host-specific evolution. Viral distribution across plant system revealed higher viral loads in stem tissue, consistent with their role in systemic transport, whereas leaves exhibited greater viral diversity, likely due to in-creased environmental exposure. The low abundance of other viral species, including Garlic viruses and Senna severe yellow mosaic virus, highlights the complex viral ecology associated with V. planifolia. This study underscores the value of metagenomic approaches for uncovering both well-characterized and novel viruses in plant systems and highlight the need for continuous viral surveillance to guide disease management strategies in economically important crops such as vanilla.

Abstract: The Polyomaviridae family contains members known for achieving high seroprevalence within their target species despite a limited genomic economy. Minimalism, by definition, allows for the clarification and streamlining of purpose via the removal of unnecessary or distracting components. Among viruses, Simian Vacuolating Virus 40 (SV40) and other polyomaviruses are master minimalists, achieving efficient replication and persistence with compact genomes of approximately 5 kb in length. This review examines how polyomaviruses employ limited genetic material and simple structure to participate in complex functions and interactions, highlighting minimalism as both an evolutionary and functional advantage. Polyomaviruses make the most of their compact genomes in each stage of the viral lifecycle through the production of multifunctional early proteins and cis-regulatory elements, utilization of alternative splicing and host infrastructure, and organization of compact structural proteins. This allows for the successful replication and proliferation of virions while also reducing evolutionary pressure and promoting host immune evasion. Examination of the implications of polyomaviral minimalism illustrates that genome economy is not a constraint, but rather a driver of biological sophistication.

Abstract: Background/Objectives: African swine fever (ASF) is a highly lethal disease of domestic pigs and wild suids that continues to cause substantial economic losses worldwide. Despite recent progress in live-attenuated ASF vaccine development, evidence supporting durable protection under repeated exposure conditions representative of endemic settings remains limited. Here, we assessed the long-term safety and protective efficacy of a live-attenuated ASFV-G-ΔI177L/ΔLVR vaccine using a repeated-challenge experimental design intended to model re-exposure in ASF-endemic regions. Methods: Vaccinated pigs were subjected to homologous virulent ASF virus challenges at multiple intervals, including repeated challenges (three sequential inoculations) and single challenges administered at 8 and 12 weeks post-vaccination. Results: Across all challenge regimens, vaccinated animals survived and remained clinically healthy, including those receiving three challenges, supporting sustained protection under repeated exposure pressure. Animals challenged at 8 or 12 weeks post-vaccination likewise exhibited complete survival, indicating maintained efficacy through at least 12 weeks. No vaccine-associated adverse clinical outcomes were detected over the study period, and post-challenge viral shedding was minimal. Conclusions: Overall, these data demonstrate that the candidate live-attenuated ASF vaccine provides excellent protective efficacy and confers sustained protection against homologous ASF virus infection. This result is expected to be equally applicable under repeated exposure conditions in regions with unstable ASF biosecurity, making it a sufficiently promising model experiment for field application in ASF epidemic areas.

Abstract: Bovine Viral Diarrhea (BVD) is an infectious disease caused by the Bovine Viral Diarrhea Virus (BVDV), a member of the genus Orthopestivirus. The disease remains endemic across Australian beef and dairy production systems, imposing a multi-million-dollar annual burden on animal health, welfare, and industry sustainability. BVDV can be transmitted both horizontally and vertically, with persistently infected (PI) animals serving as the primary source of infection. Rapid identification and subsequent culling of PI animals are fundamental requirements for any successful eradication program. Currently, Australia’s decentralised, non-compulsory approach places the responsibility of biosecurity on individual producers, resulting in a fragmented national landscape. This review proposes that the strategic deployment of rapid, field-deployable point-of-care (POC) diagnostics serves as the transformative catalyst needed for a coordinated national eradication pathway. POC approaches utilising technologies such as lateral flow assays, nucleic acid amplification tests, and biosensors enable real-time, crush-side diagnosis and high-throughput surveillance, proving effective for early detection and control of infectious diseases. When integrated with robust biosecurity measures and optimised vaccination strategies, these POC advancements offer a scientifically sound and commercially viable pathway toward the systematic eradication of BVDV in the Australian cattle industry.

Abstract: Submissions of sequences consistent with SARS-CoV-2 BA.3.2, a newly observed saltation variant, have increased in GISAID since November 2025. Saltation events are often prioritized for monitoring due to extensive divergence. Using 12-category nucleotide substitution spectra and principal component analysis, we show that BA.3.2 has a mutation-spectrum profile distinct from BA.1 and BA.2.86, lacking pronounced transversion enrichment while retaining spike-focused substitution enrichment. Unlike BA.1, BA.3.2 shows no clear enrichment of basic residue-introducing substitutions in spike.

Abstract: Glutamine is the most abundant amino acid in human plasma and tissues and plays essential roles in cellular metabolism, biosynthesis, and redox homeostasis. Beyond these canonical functions, glutamine availability and utilization have emerged as key regulators of multiple cellular stress responses, including the integrated stress response, endoplasmic reticulum stress, metabolic checkpoint signaling, and autophagy. During viral infection, host glutamine metabolism is frequently reprogrammed to meet the energetic and biosynthetic demands of viral replication, thereby inducing or reshaping glutamine-linked stress pathways. Increasing evidence indicates that these stress responses are not merely secondary consequences of infection but actively influence key stages of the viral life cycle, including viral entry, genome replication, protein synthesis, and host antiviral responses. In this review, we summarize current advances in understanding how glutamine metabolism regulates cellular stress responses in the context of both viral and non-viral infections, and how these pathways, in turn, modulate viral pathogenesis and host defense. We discuss the context-dependent roles of glutamine-linked stress signaling in either promoting viral replication or restricting infection, depending on viral species, host cell type, and metabolic conditions. Finally, we highlight emerging concepts and unresolved questions, including the potential of targeting glutamine metabolism and associated stress pathways as host-directed antiviral strategies. A deeper understanding of the interplay between glutamine metabolism, cellular stress responses, and viral infection may provide new insights into disease mechanisms and inform the development of novel therapeutic approaches.

Abstract: Percutaneous cable infection of left ventricular assist device (LVAD) patients is a significant source of morbidity, often caused by biofilm producing or multidrug resistant bacteria. We hypothesized that bacteriophage viruses can be identified from biological samples of patients with active driveline infection. Six patients with local percutaneous lead infections were enrolled. Microbiological samples were collected from the infected wound and other skin regions. The isolated viral strains and phages from wastewater samples were then tested against the pathogen bacterial cultures in vitro. Biofilm disruption assay and genetic analysis of the strains were also performed. Bacteriophages with lytic activity could be identified from samples of two patients. One patient contained four strains showing strong efficacy against his own Staphylococcus epidermidis. Furthermore, this bacterium was susceptible to phages identified from another patient and strains from wastewater samples. Genomic analysis suggested lysogenic lifestyle of the phages. However, none of them have shown any microbiological signs of lysogeny. In conclusion, we have been able to prove in vitro lytic activity of bacteriophages originating from the same LVAD patient. We also found effective phages in biological samples of other patients and wastewater samples, suggesting that patients implanted in the same center may share bacteriophage flora.

Abstract: Central Africa exhibits the highest genetic diversity of HIV-1 globally, reflecting its role as the region where the virus first emerged. A product of this diversity is the increased prevalence of drug resistance mutations (DRMs), which has been documented in the region, particularly to NRTIs and NNRTIs, highlighting the importance of studying this diversity. This has led to a significant amount of literature being produced on the topic, although it is scattered throughout multiple niche sources, investigates different populations and reports variable outcomes. This review aims to compile this information, offering pooled regional and country-level proportions of subtypes, recombinant forms and individual DRMs, exploring their evolution from 2000 to 2025. To achieve these objectives, a meta-analysis will be performed for each subtype classification and individual DRM, with a subgroup analysis using the country of origin of the samples and a meta-regression analysis using the sample collection date. Methodological quality of the studies will be addressed using a proper tool for systematic reviews of prevalence, heterogeneity using the I² statistic, and publication bias using Egger’s regression test. Finally, a bibliometric analysis will provide an overview of the research landscape in HIV-1 molecular epidemiology in Central Africa and enhance the understanding of subtype distribution and diversity hotspots.

Abstract: Angola is one of the countries with the highest HIV-1 genetic diversity, yet the implications of this diversity for antiretroviral therapy remain insufficiently characterised. Following the introduction of dolutegravir (DTG) in Angola in 2021, evaluating transmitted drug resistance prior to its widespread implementation is essential to inform treatment strategies and establish a baseline for future surveillance. In this study, 243 blood samples were collected from treatment-naïve people living with HIV attending the General Hospital of Benguela, Angola. The integrase coding region of proviral DNA was amplified and sequenced using the Sanger method. Phylogenetic relationships were inferred using a maximum likelihood approach, recombinant forms were characterised by bootscanning analysis, and resistance-associated mutations to integrase strand transfer inhibitors were identified using Stanford HIVdb, ANRS-MIE, and IAS-USA algorithms. A total of 92 integrase sequences were successfully obtained, revealing 16 distinct genetic forms, with unique recombinant forms accounting for 50.0%, followed by subtype C (10.9%) and sub-subtype F1 (8.7%). Five accessory mutations (L74I, L74M, Q95K, T97A, and E157Q) and one major mutation (E92G) were detected, corresponding to an overall prevalence of 28.4%. These findings highlight the extensive HIV-1 genetic complexity in Angola and support the continued use of DTG-based regimens, while underscoring the importance of sustained surveillance of integrase inhibitor resistance.

Abstract: The Hepatitis E virus (HEV) is a zoonotic virus of global concern that circulates in both domestic and wild pig populations. Understanding its presence and dynamics in wildlife reservoirs is crucial for assessing spillover risks and designing One Health surveillance strategies. HEV is classified into eight distinct genotypes, two of which (genotypes 3 and 4) infect both humans and swine, representing a major public health concern in Europe. This study explored the occurrence, genetic diversity, and evolutionary relationships of HEV in wild boars (Sus scrofa scrofa) from mainland Portugal. Organ samples from a total of 120 wild boar were collected from seven different districts in Portugal. HEV RNA was detected in four animals (3.3%), all of which were collected from municipalities in the Évora district, near the land border with Spain. Of the four wild boars that tested positive for HEV in the diagnostic context, it was possible to genotype and characterize one through full sequencing. Phylogenetic analysis based on the complete genome, revealed that this strain clustered with predominantly human-derived HEV-3m sequences from Spain and France, highlighting its zoonotic potential. The inclusion of 14 sequences from domestic swine in a second phylogenetic analysis performed with a small fragment allowed to conclude that sub-genotypes 3e, 3f and 3m are circulating in this population with no spatial or temporal segregation. Phylogeographic analysis suggests that there have been several transmission events between Spain and France and estimated that this HEV strain is most likely to have been introduced from Spain. The fact that these four positive samples from wild boar in south-eastern Portugal all have a common spatial origin, together with the absence of detections in the other six districts tested and the presence of known HEV hotspots in south-western Spain, supports the hypothesis that this strain in Portugal is most likely from HEV circulation in Spanish wild boar, facilitated by cross-border movement of wild boar along the south-eastern Portuguese border. Although recombination events were identified in several HEV-3 strains, none were detected in the sequence obtained in this study or in other HEV-3m strains. This study provides the first molecular evidence of HEV-3m circulation in wild boars in Portugal, offering valuable insight into the HEV strain circulation in European wildlife populations. The zoonotic potential of HEV and the likelihood of interspecies transmission highlight the need for coordinated cross-border surveillance and integrated One Health strategies.

Abstract: Human endogenous retroviruses (HERVs) are potential driving forces of the pathophysiology of Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), linking post-infectious immune dysfunction to chronic inflammation and immune and neurocognitive dysfunction that are hallmark features of ME/CFS. Accumulating evidence from related autoimmune diseases and cancers has shown that reactivated HERVs can contribute to disease pathogenesis by amplifying immune activation through viral protein-mediated innate sensing, Long Terminal Repeat (LTR)-driven transcription, and disrupting epigenetic silencing. HERV signatures are therefore promising biomarkers for diagnosis, patient stratification for drug-repurposing trials, and therapy monitoring. Accumulating evidence suggests a possible correlation between HERV expression and ME/CFS symptom severity, alterations of immune phenotypes, function and inflammatory gene networks. Importantly, locus-specific HERV profiling is a promising approach for distinguishing ME/CFS from overlapping or co-morbid conditions and healthy controls. Furthermore, HERV-targeted antibodies, immune modulators, epigenetic and antiviral interventions offer promise as concomitant therapeutic strategies for ME/CFS. Additional research incorporating viromics and other-omics validation, functional assays, and HERV-stratified clinical trials is now needed to realise this potential and to transform ME/CFS from a symptom-based syndrome into a mechanism-driven, treatable condition.

Abstract: Schmallenberg virus (SBV) has evolved from an emergent Orthobunyavirus identified in Europe in 2011 into an endemic pathogen with complex epidemiological dynamics. This review synthesizes advances in SBV research over the past 15 years, focusing on molecular pathogenesis, transmission ecology, and control strategies. We highlight the NSs protein as a key interferon antagonist, the mechanisms underlying fetal neurotropism, and the influence of climate change on Culicoides vector biology. Recent evidence of winter vector activity challenges traditional assumptions of transmission seasonality, with implications for disease management and trade regulations. Advances in diagnostics and vaccine development—including DIVA‑compatible and mRNA platforms—are critically evaluated, highlighting progress and persistent implementation gaps. Finally, we propose SBV as a model system for understanding arbovirus persistence and emergence in temperate regions within a One Health framework.

Abstract: High-throughput sequencing (HTS) was applied to investigate the virome of European beech (Fagus sylvatica) from asymptomatic leaves and leaves exhibiting chlorosis, line patterns and malformation. Total RNA extracted from six samples, including herbarium material collected in 1967 and 1968 and contemporary samples from France, Germany, and the Netherlands, was subjected to Illumina sequencing followed by de novo assembly, sequence similarity searches and phylogenetic analyses. In each sample, contigs belonging to a previously undescribed virus within the genus Carlavirus were obtained. The virus was tentatively named beech carlavirus. The detection of the virus over more than five decades and in three European countries indicates its long-term and a probable wider occurrence and circulation. Moreover, its prolonged unnoticed presence suggests that it does not induce noticeable and acute disease outbreaks. These findings underscore the value of integrating historical and recent field samples through collaborative data sharing to improve insight into virus diversity and ecology in forest trees.

Abstract: Madagascar experienced a severe measles epidemic between September 2018 and mid-2019, resulting in over 146,000 cases and 1,200 deaths, primarily among children under 15. This epidemic occurred in a context of low vaccination coverage. Prior to this epidemic, no genotyping data for the measles virus (MeV) or rubella virus (RuV) were available for Madagascar. This study aimed to molecularly characterize MeV and RuV strains circulating during the epidemic. A total of 310 biological samples (gingival swabs, urine, and stool) were collected from 288 suspected patients with a mean age of 11.4 years. Viral detection was performed by real-time RT-PCR, followed by conventional RT-PCR and sequencing for genotyping of the N and H genes of MeV and the E1 gene of RuV. The results revealed co-circulation of the two viruses, with detection rates of 39.9% (115/288) for MeV and 40.0% (70/175) for RuV. The mean age differed significantly between MeV-positive (12.0 years) and RuV-positive (7.2 years) patients, with 35.1% and 43.8% of cases occurring in children under five years of age, respectively. Phylogenetic analysis identified all MeV strains as belonging to genotype B3, showing high similarity to strains circulating globally in 2018-2019, suggesting recent importation. Meanwhile, the phylogenetic profile of RuV strains, all belonging to genotype 2B and displaying greater genetic diversity, was characteristic of endemic rubella in a partially vaccinated or unvaccinated population. This study provides the first genotyping data for Madagascar, essential for monitoring virus circulation and supporting elimination efforts in the African region.

Abstract: Bluetongue virus (BTV), the prototype orbivirus, infects livestock, causing high morbidity and mortality and impacting global trade. BTV is a non-enveloped, double-capsid virus, composed of seven structural proteins and a genome of ten double-stranded RNA segments. This manuscript highlights our recent findings on the molecular and structural mechanisms underlying BTV entry and assembly during replication. Viral entry is a stepwise, pH-dependent process. The virus-neutralisation, outermost protein VP2 attaches to sialic acids and senses the acidic pH of early endosomes, triggering its dissociation. Subsequently, the second outer capsid protein, VP5, undergoes major changes in late endosomes, forming a membrane-penetrating pore that releases the transcriptionally-active inner core into the host cytoplasm. Core assembly also proceeds stepwise and requires accurate packaging of ten positive-sense RNA segments. These segments form an RNA–RNA interaction network independent of viral proteins, beginning with the smaller segments and guiding complete genome assortment. The small capsid protein VP6, interacts with VP3 to facilitate RNA encapsidation. While infectious cores assemble in-vitro without non-structural proteins, NS2 is essential for the in-vivo formation of viral inclusion bodies via liquid–liquid phase separation, concentrating viral components and promoting genome assembly. These comprehensive characterisations of BTV provide future control strategies for related reoviruses.

Abstract: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide, with zoonotic genotypes detected in humans and animals. In Africa, limited data exist on environmental HEV circulation. Here, we report the first detection of Rocahepevirus ratti (RHEV) in urban wastewater from Conakry, Guinea. From December 2024 to April 2025, Rocahepevirus ratti (RHEV) has been detected in 35 out of 180 urban untreated wastewater samples in Conakry, Guinea. Phylogenetic analysis of partial HEV ORF1 genome segments reveals clustering with African rodents RHEV strains, highlighting environmental contamination and potential zoonotic risk for human population in proximity. This finding underscores the need for integrated One Health surveillance to monitor HEV transmission at the human-animal-environment interface in West Africa particularly in Guinea.

Abstract: Enterovirus alphacoxsackie (EV-A) is a highly diverse viral species containing at least 25 serotypes of viruses which show distinct infectivity and pathogenicity. Increasing studies show that EV-A protease 2A plays critical roles in virus-host interactions. Although 2A is usually considered as a non-structural protein highly conserved among different EV-A serotypes, its orthologs of different EV-A serotypes harbor abundant single amino acid polymorphisms (SAPs), probably contributing to the variance of infectivity and patho-genicity of EV-As. However, the SAP profile of EV-A 2A and its functional impacts have been poorly understood, mainly due to the unequal contribution to protein function of dif-ferent SAP sites. Herein, we developed Single Amino Acid Polymorphism Statistics (SAAPS), an information-entropy-based algorithmic pipeline, to identify key SAP sites (kSAPs) related to functional variance of EV-A 2A. As the result, we identified 56 kSAPs from 2A of 25 EV-A serotypes. Based on the kSAPs, the 2As can be clustered into three major groups with a few outliers, which was distinct from the clustering generated by phylogenetic analysis using the whole amino acid sequences. Functional verification with transcriptomic profiles of HEK-293T cells expressing different 2A variants revealed better phenotypic matching of kSAP-based clustering than that of phylogenetic clustering. No-tably, EV-A89, an outlier identified by kSAP clustering but not phylogenetic clustering, showed unique expressing and self-cleavage patterns which were not observed in other 2As. These findings demonstrated the good performance of SAAPS and functional SAP profile of EVA 2As, contributing to the understanding of the variance of EV-A infectivity and pathogenicity.