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Microbes & Immunity SARS-CoV-2 complementary classification

transmissibility, and immune escape mechanisms. Genetic ambiguous bases and incomplete sequences removed
divergence thresholds were established by analyzing the before analysis. The final alignment contained a total
evolutionary distances between recognized subtypes of 2,214 nucleotide positions, with discrete clustering
or genotypes of HIV-1, HCV, and influenza A virus, as observed in the ML phylogenetic analysis (Figure 2).
outlined in the sections above. The evolutionary divergence between subtypes was

A review of the literature was conducted in PubMed/ assessed using pairwise genetic distances (Table 2). The
MEDLINE to identify key functional parameters that have genetic distances among HCV subtypes ranged from
historically defined viral lineages in other RNA viruses, 0.371 s/s (1a vs. 1b) to 0.674 s/s (3a vs. 4a), reflecting the
including HIV-1, HCV, influenza A virus, and previous well-documented high genetic diversity of HCV. The mean
SARS-CoV-2 designated as VOCs. The exact search strategy, divergence across all subtype comparisons was 0.57 s/s,
concluded on 31 January 2025, was: ((“SARS-CoV-2” OR which is notably higher than that observed in HIV-1 env
“COVID-19” OR “HIV-1” OR “Hepatitis C Virus” OR sequences.
“HCV” OR “Influenza A”) AND (“variant of concern”
OR “VOC” OR “subtype” OR “genotype” OR “lineage” 3.3. Influenza A virus dataset and evolutionary
OR “clade”) AND (“transmissibility” OR “replication distance analysis
fitness” OR “R0” OR “immune escape” OR “neutralizing The influenza dataset included 49 full-length HA
antibodies” OR “vaccine resistance” OR “vaccine escape” sequences, representing two major subtypes, H1 and H3,
OR “clinical severity” OR “hospitalization” OR “mortality” as indicated in the ML phylogenetic tree (Figure 3). The
OR “epidemiological dominance” OR “variant persistence” genetic divergence between these subtypes was estimated at
OR “lineage replacement”)). 1.956 s/s, which is substantially higher than the divergence
Criteria were categorized into four virologically relevant observed in both HIV-1 and HCV subtypes.
domains: (1) Transmissibility: Evidence of increased 3.4. SARS-CoV-2 dataset and evolutionary distance
replication fitness 40,91,108-112 ; (2) Immune escape potential: analysis
≥2-fold reduction in neutralizing antibody effectiveness,
impacting vaccine efficacy, or convalescent immunity 92,113 ; The SARS-CoV-2 S sequence dataset in the ML phylogenetic
(3) Clinical impact: Increased hospitalization, mortality, or analysis showed a clear separation into five different
multisystem complications compared to prior circulating statistically supported monophyletic clades (Figure 4).
variants 114-119 ; and (4) Epidemiological dominance: Variant Pairwise genetic distance analysis of SARS-CoV-2 variants
must persist for >12 months, indicating sustained selective revealed minimal divergence, with nucleotide substitution
advantage. 89,120-122 rates ranging from 0.002 to 0.006 s/s. The highest genetic
distance was observed between Omicron and Beta
3. Results (0.006 s/s) and Omicron and Alpha (0.006 s/s), while the
lowest was between Beta and Gamma (0.002 s/s). Variants
3.1. HIV-1 env dataset description and ML Delta, Beta, and Gamma exhibited similar divergence levels
phylogenetic analysis relative to Alpha (0.003 – 0.004 s/s), indicating limited
A total of 58 full-length HIV-1 env sequences were analyzed, evolutionary separation. These findings demonstrate that
representing six major subtypes and CRFs. The ML SARS-CoV-2 variants remain within a constrained genetic
phylogenetic tree robustly supported the classification of space, with divergence levels substantially lower than those
these subtypes into distinct lineages, with bootstrap values observed in HIV-1 subtypes, HCV genotypes, or influenza
exceeding 0.99, indicating high confidence in the inferred A virus subtypes (Table 3).
evolutionary relationships (Figure 1). The distances ranged
from 0.157 s/s (A vs. CRF02_AG) to 0.235 s/s (D vs. 3.5. Establishing the genetic divergence threshold
CRF01_AE), with an overall mean divergence of 0.21 s/s. based on sequences analyzed
The highest divergence was observed between subtypes D To objectively classify viral genetic divergence, three
and CRF01_AE (0.235 s/s), whereas the lowest was found evolutionary divergence thresholds were established
between subtypes A and CRF02_AG (0.157 s/s) (Table 1). based on the minimum observed genetic distances in
HIV-1, HCV, and influenza A virus. Three thresholds,
3.2. HCV E1 dataset and evolutionary distance shown in Figure 5, are as follows: low divergence, below
estimation 0.157 s/s (based on HIV-1 subtype minimum); moderate
A total of 40 HCV E1 sequences were analyzed, representing divergence, between 0.157 and 0.371 s/s (based on HCV
four major subtypes: 1a, 1b, 3a, and 4a. The dataset was subtype minimum); and high divergence, above 0.371 s/s
curated to ensure full-length coding sequences, with all (with influenza A virus defining the upper bound at

Volume 2 Issue 3 (2025) 92 doi: 10.36922/MI025190042