Comparative Analysis of SARS-CoV-2 Variants of Concern, Including Omicron, Highlights Their Common and Distinctive Amino Acid Substitution Patterns, Especially at the Spike ORF.

In order to gain a deeper understanding of the recently emerged and highly divergent Omicron variant of concern (VoC), a study of amino acid substitution (AAS) patterns was performed and compared with those of the other four successful variants of concern (Alpha, Beta, Gamma, Delta) and one closely related variant of interest (VoI-Lambda). The Spike ORF consistently emerges as an AAS hotspot in all six lineages, but in Omicron this enrichment is significantly higher. The progenitors of each of these VoC/VoI lineages underwent positive selection in the Spike ORF. However, once they were established, their Spike ORFs have been undergoing purifying selection, despite the application of global vaccination schemes from 2021 onwards. Our analyses reject the hypothesis that the heavily mutated receptor binding domain (RBD) of the Omicron Spike was introduced via recombination from another closely related Sarbecovirus. Thus, successive point mutations appear as the most parsimonious scenario. Intriguingly, in each of the six lineages, we observed a significant number of AAS wherein the new residue is not present at any homologous site among the other known Sarbecoviruses. Such AAS should be further investigated as potential adaptations to the human host. By studying the phylogenetic distribution of AAS shared between the six lineages, we observed that the Omicron (BA.1) lineage had the highest number (8/10) of recurrent mutations.

The Remarkable Evolutionary Plasticity of Coronaviruses by Mutation and Recombination: Insights for the COVID-19 Pandemic and the Future Evolutionary Paths of SARS-CoV-2.

Coronaviruses (CoVs) constitute a large and diverse subfamily of positive-sense single-stranded RNA viruses. They are found in many mammals and birds and have great importance for the health of humans and farm animals. The current SARS-CoV-2 pandemic, as well as many previous epidemics in humans that were of zoonotic origin, highlights the importance of studying the evolution of the entire CoV subfamily in order to understand how novel strains emerge and which molecular processes affect their adaptation, transmissibility, host/tissue tropism, and patho non-homologous genicity. In this review, we focus on studies over the last two years that reveal the impact of point mutations, insertions/deletions, and intratypic/intertypic homologous and non-homologous recombination events on the evolution of CoVs. We discuss whether the next generations of CoV vaccines should be directed against other CoV proteins in addition to or instead of spike. Based on the observed patterns of molecular evolution for the entire subfamily, we discuss five scenarios for the future evolutionary path of SARS-CoV-2 and the COVID-19 pandemic. Finally, within this evolutionary context, we discuss the recently emerged Omicron (B.1.1.529) VoC.

The Neighborhood of the Spike Gene Is a Hotspot for Modular Intertypic Homologous and Nonhomologous Recombination in Coronavirus Genomes.

Coronaviruses (CoVs) have very large RNA viral genomes with a distinct genomic architecture of core and accessory open reading frames (ORFs). It is of utmost importance to understand their patterns and limits of homologous and nonhomologous recombination, because such events may affect the emergence of novel CoV strains, alter their host range, infection rate, tissue tropism pathogenicity, and their ability to escape vaccination programs. Intratypic recombination among closely related CoVs of the same subgenus has often been reported; however, the patterns and limits of genomic exchange between more distantly related CoV lineages (intertypic recombination) need further investigation. Here, we report computational/evolutionary analyses that clearly demonstrate a substantial ability for CoVs of different subgenera to recombine. Furthermore, we show that CoVs can obtain-through nonhomologous recombination-accessory ORFs from core ORFs, exchange accessory ORFs with different CoV genera, with other viruses (i.e., toroviruses, influenza C/D, reoviruses, rotaviruses, astroviruses) and even with hosts. Intriguingly, most of these radical events result from double crossovers surrounding the Spike ORF, thus highlighting both the instability and mobile nature of this genomic region. Although many such events have often occurred during the evolution of various CoVs, the genomic architecture of the relatively young SARS-CoV/SARS-CoV-2 lineage so far appears to be stable.

Mutation Profile of HPV16 L1 and L2 Genes in Different Geographic Areas.

The causal relationship between HPV and cervical cancer in association with the high prevalence of high risk HPV genotypes led to the design of HPV vaccines based on the major capsid L1 protein. In recent years, capsid protein L2 has also become a focal point in the field of vaccine research. The present review focuses on the variability of HPV16 L1 and L2 genes, emphasizing the distribution of specific amino acid changes in the epitopes of capsid proteins. Moreover, a substantial bioinformatics analysis was conducted to describe the worldwide distribution of amino acid substitutions throughout HPV16 L1, L2 proteins. Five amino acid changes (T176N, N181T; EF loop), (T266A; FG loop), (T353P, T389S; HI loop) are frequently observed in the L1 hypervariable surface loops, while two amino acid substitutions (D43E, S122P) are adjacent to L2 specific epitopes. These changes have a high prevalence in certain geographic regions. The present review suggests that the extensive analysis of the amino acid substitutions in the HPV16 L1 immunodominant loops may provide insights concerning the ability of the virus in evading host immune response in certain populations. The genetic variability of the HPV16 L1 and L2 epitopes should be extensively analyzed in a given population.

Large-scale genomic analysis reveals recurrent patterns of intertypic recombination in human enteroviruses.

Recombination is a driving force for the emergence, evolution and virulence/epidemics of viruses, comprising the Enterovirus genus of the Picornaviridae family, important for human and animal health. By analyzing 2949 complete genomes/coding sequences, we provide a thorough and up-to-date overview of the genome-wide patterns and hotspots of intertypic recombination between the genogroups of this genus. Two prominent recombination hotspots are identified/verified, at the 5'UTR-capsid region junction, and at the beginning of the P2 region. In general, P2 was enriched in recombination events. Key phylogenetic groups implicated in recombination events are E71 and CVA6 in Enterovirus A species, E30 and E6 in Enterovirus B species, polioviruses 1 and 2 in Enterovirus C species. In addition, many events involve recombination partners that have not been sequenced yet, thus strongly suggesting a large environmental reservoir of genetic variation with a high potential for the emergence of new modified pathogens by recombination.

Association of p16 (CDKN2A) polymorphisms with the development of HPV16-related precancerous lesions and cervical cancer in the Greek population.

The tumor suppressor protein p16 plays a fundamental role in cell cycle regulation and exerts a protective effect against tumor growth. Two different polymorphisms at positions 540 and 580 at the 3'UTR of exon 3 of p16 gene are implicated in several types of cancer, while their role in cervical cancer development remains rather vague. In the present study, we investigated for the impact of p16 genotypes/haplotypes on patients' vulnerability to cervical disease and examined whether these factors can be used as progression markers in the Greek population. A total of 96 HPV16 positive samples and histologically confirmed as LSIL (42 samples), HSIL (44 samples), and cervical cancer cases (10 samples) along with 50 control cases were tested. The identification of p16 polymorphisms was performed by PCR-RFLP methodology. The present analysis revealed that women with p16 540 CG/GG genotype are at a 2.7-fold higher risk of developing HPV16-associated HSIL (OR = 2.7, 95%CI: 1.01-6.6, P = 0.028). The G allele can be regarded as a risk factor of developing HSIL in the Greek population (OR = 2.7, 95%CI: 1.2-5.9, P = 0.012). Moreover, p16 polymorphism C580T is not associated with the growth of cervical lesion in Greek patients, while 540G/580C haplotype can be regarded as a risk haplotype of developing HSIL (OR = 3.67, 95%CI: 1.56-8.6, P = 0.0019). Our results demonstrated that p16 C540G polymorphism influence patients' susceptibility to more severe dysplasia and consequently this polymorphism could potentially emerge as a valuable biomarker for HSIL development in the Greek population.

Intra- and inter-serotypic recombinations in the 5΄ UTR-VP4 region of Echovirus 30 strains.

Recombination has been recognized as a major mechanism of evolution in enteroviruses. The Echovirus 30 (E-30) strain Gior was sequenced and phylogenetically compared to all available E-30 sequences to detect recombination events between the 5΄UTR and VP1 genomic regions. The comparison of phylogenetic trees of the 5΄UTR and VP1 revealed incongruences concerning strains, lineages and sub-lineages. Comparative analysis of 62 E-30 sub-genomic sequences revealed six different recombination events that almost all occurred in the same region, having a start point in the 3΄end of the 5΄ UTR and end point in VP4. The only exception was the sub-lineage of Gior for which both borders of recombination were located in the 5΄UTR. These results describe for the first time recombination events in this region in circulating EV-B strains, revealing the exact points of these recombination events, highlighting the impact of such events on the evolution and epidemiology of enteroviruses.

Association of codon 72 polymorphism of p53 with the severity of cervical dysplasia, E6-T350G and HPV16 variant lineages in HPV16-infected women.

Purpose. Polymorphic variability in the tumour-suppressor protein p53 at codon 72 has a considerable impact on cervical cancer development. The present study clarified the association between p53 codon 72 genotypes and the risk of cervical disease in Greek patients. We also examined whether the presence of specific p53 genotypes in combination with HPV16 variants or E6 T350G sequence variation can modify an individual's susceptibility to cervical disease.Methodology. The analysis of p53 genotypes was performed through PCR-RFLP. Sequence and phylogenetic tree analyses of the HPV16 E6 gene were also performed in order to identify HPV16 variants and T350G sequence variation.Results/Key findings. The outcomes of the present analysis revealed that women who are homozygous for the arg genotype are at a 4.17-fold higher risk of developing HPV16-associated HSIL+ (OR=4.17, 95 % CI:1.48-4.9, P=0.0049). Moreover, p53 arg/arg patients infected by an HPV16 prototype strain were associated with an increased risk of more severe lesions, while a significant relationship between the p53 arg/arg genotype in patients with T350G sequence variation and the risk of high-grade squamous intraepithelial lesions (HSILs) was revealed.Conclusion. The oncogenic potential of the virus is increased by the presence of the p53 arg/arg genotype in the Greek population in such a way that the specific protein interaction E6 (L83V)-p53 (Arg-72) can modify an individual's susceptibility to cervical disease.

T-RECs: rapid and large-scale detection of recombination events among different evolutionary lineages of viral genomes.

BACKGROUND: Many computational tools that detect recombination in viruses are not adapted for the ongoing genomic revolution. A computational tool is needed, that will rapidly scan hundreds/thousands of genomes or sequence fragments and detect candidate recombination events that may later be further analyzed with more sensitive and specialized methods. RESULTS: T-RECs, a Windows based graphical tool, employs pairwise alignment of sliding windows and can perform (i) genotyping, (ii) clustering of new genomes, (iii) detect recent recombination events among different evolutionary lineages, (iv) manual inspection of detected recombination events by similarity plots and (v) annotation of genomic regions. CONCLUSIONS: T-RECs is very effective, as demonstrated by an analysis of 555 Norovirus complete genomes and 2500 sequence fragments, where a recombination hotspot was identified at the ORF1-ORF2 junction.

Serum Neutralization Assay for the Determination of Antibody Levels Against Non-Polio Enterovirus Strains in Central and Western Greece.

Mutations and recombination events have been identified in enteroviruses. Point mutations accumulate with a frequency of 6.3 × 10(-4) per base pair per replication cycle affecting the fitness, the circulation, and the infectivity of enteroviral strains. In the present report, the serological status of the Central and Western Greek population (Larissa and Ioannina, respectively) in the 1-10-year, 11-20-year, 21-30-year, and 31-40-year age groups against six non-polio enterovirus strains, their respective echovirus prototypes, and Sabin 1, 2, and 3 vaccine strains was evaluated, through serum-neutralization assay. In the Western Greek population, antibody levels were detected only for clinical isolates of E30 serotype in all age groups, and for environmental isolate LR61G3 (E6 serotype) only in the 31-40 age group, whereas an immunity level was observed in the Central Greek population, against all strains, except for EIS6B (E3 serotype). Amino acid substitutions were encountered across the structural region of the capsid, between the prototypes and the respective isolates. These substitutions may alter the antigenicity of each strain and may explain the variations observed in the neutralization titers of the different strains. As a consequence, these substitutions severely affect antibody binding and increase the ability of the virus to escape the immune response. It is tempting to assume that changes in the antigenic properties observed in circulating echoviruses represent a selection of viral variants that are less prone to be neutralized by human antibodies. These facts argue for the need of immunological studies to the population to avoid epidemics due to the circulation of highly evolved derivatives.

Molecular epidemiology and evolutionary dynamics of Echovirus 3 serotype.

Echovirus 3 (E3) serotype has been related with several neurologic diseases, although it constitutes one of the rarely isolated serotypes, with no report of epidemics in Europe. The aim of the present study was to provide insights into the molecular epidemiology and evolution of this enterovirus serotype, while an E3 strain was isolated from sewage in Greece, four years after the initial isolation of the only reported E3 strain in the same geographical region. Phylogenetic analysis of the complete VP1 genomic region of that E3 strain and of those available in GenBank suggested three main genogroups that were further subdivided into seven subgenogroups. Further evolutionary analysis suggested that VP1 genomic region of E3 was dominated by purifying selection, as the vast majority of genetic diversity presumably occurred through synonymous nucleotide substitutions and the substitution rate for complete and partial VP1 sequences was calculated to be 8.13×10(-3) and 7.72×10(-3) substitutions/site/year respectively. The partial VP1 sequence analysis revealed the composite epidemiology of this serotype, as the strains of the three genogroups presented different epidemiological characteristics.

Recombination among human non-polio enteroviruses: implications for epidemiology and evolution.

Human enteroviruses (EV) belong to the Picornaviridae family and are among the most common viruses infecting humans. They consist of up to 100 immunologically and genetically distinct types: polioviruses, coxsackieviruses A and B, echoviruses, and the more recently characterized 43 EV types. Frequent recombinations and mutations in enteroviruses have been recognized as the main mechanisms for the observed high rate of evolution, thus enabling them to rapidly respond and adapt to new environmental challenges. The first signs of genetic exchanges between enteroviruses came from polioviruses many years ago, and since then recombination has been recognized, along with mutations, as the main cause for reversion of vaccine strains to neurovirulence. More recently, non-polio enteroviruses became the focus of many studies, where recombination was recognized as a frequent event and was correlated with the appearance of new enterovirus lineages and types. The accumulation of multiple inter- and intra-typic recombination events could also explain the series of successive emergences and disappearances of specific enterovirus types that could in turn explain the epidemic profile of circulation of several types. This review focuses on recombination among human non-polio enteroviruses from all four species (EV-A, EV-B, EV-C, and EV-D) and discusses the recombination effects on enterovirus epidemiology and evolution.

Determination of human papillomavirus 16 physical status through E1/E6 and E2/E6 ratio analysis.

Human papillomavirus (HPV) 16 genome integration into the host chromosome is a crucial event during the life cycle of the virus and a major step towards carcinogenesis. The integration of HPV16 DNA promotes a constitutive high expression level of E6 and E7 oncoproteins, resulting in the extensive proliferation of the infected epithelial cells. In the present report the physical status of the HPV16 genome was studied, through determination of E1/E6 and E2/E6 DNA copy number ratios in 61 cervical samples of low- and high-grade malignancy and 8 cervical cancer samples, all of them associated with HPV16 infection. The selection of E1, E2 and E6 amplification target regions was performed according to the most prevalent deleted/disrupted sites of E1 and E2 genes. For this target selection we also considered the most conserved regions of E1, E2 and E6 genes among the same HPV16 isolates that were recently reported by our group. The analysis of HPV16 DNA form revealed a significant association among the mixed DNA forms in low-grade and high-grade malignancies, (χ(2), P<0.01). The comparative analysis of E1/E6 and E2/E6 in the same cervical samples provides an accurate picture of HPV16 DNA form and may reveal whether different HPV16 DNA integrants coexist in the same cervical sample or not. This study proposes that E1/E6 and E2/E6 ratios determine with accuracy the HPV16 DNA integration pattern and may predict multiple integration events in the examined sample, thus providing significant information about the progression of cervical dysplasia.

Combined 5' UTR RFLP analysis and VP1 sequencing for epidemic investigation of enteroviruses.

Enteroviruses, the main cause of aseptic meningitis, consist of 100 serotypes, and many of them have been associated with large outbreaks. In the present study, a comparison of RFLP analysis of the 5' untranslated region (5'UTR) and sequencing of both the 5'UTR and VP1 regions was conducted for epidemiological linkage of 27 clinical enterovirus strains. The clinical enterovirus strains were clustered into five restriction profile groups. Even though the restriction profile clusters of clinical isolates were not related to those of the respective prototype strains, epidemiological relationships between the members of each cluster were observed. The restriction profile clusters in the 5'UTR corresponded to the phylogenetic clusters in the VP1 genomic region. The incongruence between the topology of Gior strain in 5'UTR and VP1 phylogenetic trees indicates a recombination event. The proposed RFLP assay in combination with VP1 sequencing can offer crucial epidemiological information about the circulating enteroviruses.

Diagnosis of Upper and Lower Respiratory Tract Bacterial Infections with the Use of Multiplex PCR Assays.

The investigation of respiratory infections by molecular techniques provides important information about the epidemiology of respiratory disease, especially during the post-vaccination era. The objective of the present study was the detection of bacterial pathogens directly in clinical samples from patients with upper and lower respiratory tract infections using multiplex polymerase chain reaction (PCR) assays developed in our laboratory. Clinical samples taken over a three-year period (2007-2009) and obtained from 349 patients (adults (n = 66); children (n = 283)) with signs and symptoms of certain upper or lower respiratory tract infections, consisted of: bronchoalveolar lavages (BAL, n = 83), pleural fluids (n = 29), and middle-ear aspirates (n = 237). Overall, 212 samples (61%) were confirmed by culture and/or PCR. Among the positive samples, Streptococcus pneumoniae (mainly serotype 3) was predominant (104/212; 49.0%), followed by non-typable Haemophilus influenzae (NTHi) 59/212; 27.8%) and Streptococcus pyogenes (47/212; 22%). Haemophilus influenzae type b was detected in only three samples. The underlying microbiology of respiratory infections is gradually changing in response to various selective pressures, such as vaccine use and antibiotic consumption. The application of multiplex PCR (mPCR) assays is particularly useful since it successfully identified the microorganisms implicated in acute otitis media or lower respiratory tract infections in nearly 75% of patients with a positive result compared to conventional cultures. Non-culture identification of the implicated pneumococcal serotypes is also an important issue for monitoring pneumococcal infections in the era of conjugate pneumococcal vaccines.

Risks associated with the use of live-attenuated vaccine poliovirus strains and the strategies for control and eradication of paralytic poliomyelitis.

The Global Polio Eradication Initiative was launched in 1988 with the aim to eliminate paralytic poliomyelitis. Two effective vaccines are available: inactivated polio vaccine (IPV) and oral polio vaccine (OPV). Since 1964, OPV has been used instead of IPV in most countries due to several economic and biological advantages. However, in rare cases, the live-attenuated Sabin strains of OPV revert to neurovirulence and cause vaccine-associated paralytic poliomyelitis in vaccinees or lead to emergence of vaccine-derived poliovirus strains. Attenuating mutations and recombination events have been associated with the reversion of vaccine strains to neurovirulence. The substitution of OPV with an improved new-generation IPV and the availability of new specific drugs against polioviruses are considered as future strategies for outbreak control and the eradication of paralytic poliomyelitis worldwide.

Genome analysis of two type 6 echovirus (E6) strains recovered from sewage specimens in Greece in 2006.

Echovirus 6 (E6) is one of the main enteroviral serotypes that was isolated from cases of aseptic meningitis and encephalitis during the last years in Greece. Two E6 (LR51A5 and LR61G3) were isolated from the sewage treatment plant unit in Larissa, Greece, in May 2006, 1 year before their characterization from aseptic meningitis cases. The two isolates were initially found to be intra-serotypic recombinants in the genomic region VP1, a finding that initiated a full genome sequence analysis. In the present study, nucleotide, amino acid, and phylogenetic analyses for all genomic regions were conducted. For the detection of recombination events, Simplot and bootscan analyses were carried out. The continuous phylogenetic relationship in 2C-3D genomic region of strains LR51A5 and LR61G3 with E30 isolated in France in 2002-2005 indicated that the two strains were recombinants. SimPlot and Bootscan analyses confirmed that LR51A5 and LR61G3 carry an inter-serotypic recombination in the 2C genomic region. The present study provide evidence that recombination events occurred in the regions VP1 (intraserotypic) and non-capsid (interserotypic) during the evolution of LR51A5 and LR61G3, supporting the statement that the genomes of circulating enteroviruses are a mosaic of genomic regions of viral strains of the same or different serotypes. In conclusion, full genome sequence analysis of circulating enteroviral strains is a prerequisite to understand the complexity of enterovirus evolution.

Determination of antigenic properties of vaccine derived poliovirus strains.

In this study, the immunity level of the southern Greek population in the 1-10-year, 11-20-year, 21-30-year and 31-40-year age groups with regard to Sabin vaccine strains and a collection of 11 recombinant and three non-recombinant poliovirus vaccine strains was determined. The results showed the lowest neutralization titre in the 21-30-year-age group against poliovirus type 3. Moreover, the capsid coding region of OPV (oral poliovirus vaccine) derivatives was sequenced in order to identify mutations that might lead to antigenic changes. In Sabin-1 derivatives a tendency of accumulation of mutations was observed in or near antigenic sites while in Sabin-2 and Sabin-3 derivatives in sites known to be involved in restoring neurovirulence or eliminating their temperature-sensitive phenotype. It was concluded that the combination of mutations in the capsid coding region and not the number of specific mutations in antigenic sites determines the antigenic properties of OPV derivatives and their reactivity with human sera.

Correlation between recombination junctions and RNA secondary structure elements in poliovirus Sabin strains.

In order to test the hypothesis that RNA structural elements promote the distribution of certain types of recombination junctions in each one of the 2C and 3D poliovirus genomic regions (Sabin 3/Sabin 2 or Sabin 1 in 2C and Sabin 2/Sabin 1 or Sabin 3 in 3D), we searched in 2C and 3D regions of reference Sabin strains for high probability RNA structural elements that could promote recombination. Recombination junctions that were identified in clinical strains of this study, as well as in clinical strains of previous studies, were superimposed on RNA secondary structure models of 2C and 3D genomic regions. Furthermore, we created an in vitro model, based on double infection of cell-culture with two poliovirus strains, for the production and identification of recombinant Sabin strains in 2C and 3D regions. Our intention was to compare the results that refer to the correlation of recombination junctions and RNA secondary structures in 2C and 3D regions of clinical strains, with the respective results of the in vitro model. Most of the recombination junctions of the clinical strains were correlated with RNA secondary structure elements, which were identical between recombining Sabin strains, and also presented high predictive value. In consensus were, the respective results originated from the in vitro model. We propose that the distribution of specific types of recombination junctions in certain regions of Sabin strains is not fortuitous and is correlated with RNA secondary structure elements identical to both recombination partners. Furthermore, results of this study highlight an important role for the stem region of the RNA structure elements in promoting recombination.