Advanced Hepatitis Management: Precision Medicine Integration

2.1 Interferons

IFNs were among the first antiviral therapies introduced for hepatitis treatment, particularly for chronic HBV and HCV [14, 17]. These naturally occurring cytokines exhibit strong antiviral, antiproliferative, and immunomodulatory properties. Specifically, IFN-α stimulates antiviral pathways through IFN-stimulated genes and directly inhibits hepatitis viruses by suppressing viral DNA synthesis, disrupting RNA-containing core particles, accelerating their decay, and degrading pre-genomic RNA and the epigenetic of covalently closed circular DNA (cccDNA) [18, 19]. This antiviral action is vital for infection control, enhancing innate immunity and potentially activating natural killer (NK) cells, which may help restore adaptive immune function [20]. As a result, IFN-α treatment can lower viral load and HBV surface antigen (HBsAg) levels and improve CD8 T-cell responses in sustained responders’ post-treatment [21].

Standard IFN therapy had a short half-life and a modest response rate of approximately 16%, often necessitating prolonged treatment durations and frequent dosing regimens [22]. These limitations are influenced by baseline factors, such as viral genotype, viral load, sex, age, geographic location, HBsAg, and alanine aminotransferase (ALT) levels [23, 24], but their predictive value for individual patients is limited. Interestingly, research on genetic polymorphisms has yielded promising insights into individual susceptibility to IFN-α treatment. Unlike HCV, interleukin 28B (IL-28B) gene polymorphism has not been shown to impact IFN-α response in chronic HBV or hepatitis D [25, 26, 27]. While certain human leukocyte antigens (HLA) polymorphisms have been linked to HBV pathogenesis in specific populations [28], their correlation with IFN-α response remains inconsistent [29]. Other genetic biomarkers, such as the rs3746662 polymorphism in adenosine deaminase acting on double-stranded RNA1 (ADAR1) and the rs7574865 polymorphism in signal transducer and activator of transcription 4 (STAT4), have shown potential as predictors of IFN-α treatment response [30, 31]. Further study is needed to clarify the link between genetic polymorphisms and IFN response, as conclusive evidence for clinical application remains limited.

Pegylated IFN-α (Peg-IFNα) formulations have emerged as a preferred treatment for chronic hepatitis due to their extended half-life, allowing for less frequent dosing and improved patient adherence [15]. Pegylation has demonstrated effectiveness in both HBV e-antigen (HBeAg)-positive and HBeAg-negative patients, achieving significant rates of viral suppression, HBeAg seroconversion, and sustained response [32, 33]. For instance, a 48-week course of Peg-IFNα treatment in HBeAg-positive liver cirrhosis patients results in an ALT normalization, a 27% reduction in HBeAg levels, and a 25% decrease in HBV DNA [34, 35, 36]. For HBeAg-negative patients, Peg-IFNα can sustain long-term suppression of HBsAg levels for up to 3 years post-treatment, reflecting a significant SVR [37]. Despite these advances, IFN-based therapies are limited by poor patient tolerance, with notable adverse effects, such as flu-like symptoms, depression, hematological abnormalities, and thyroid dysfunction. These issues can lead to a high rate of treatment discontinuation and contraindicate Peg-IFNα use in patients with decompensated or severe compensated cirrhosis and during pregnancy [3, 24].

Ribavirin, an oral guanosine analog, was introduced to enhance interferon (IFN) therapy, leading to reduced relapse rates and modest improvements in cure rates, reaching 41% for genotype 1 and nearly 75% for other genotypes [38]. It remained the standard therapy until 2011, though its effectiveness varied, showing greater success in HBeAg-positive patients, hindered by significant toxicity, especially in advanced liver disease [39]. For severe cases or acute-on-chronic liver failure caused by the hepatitis E virus, ribavirin is recommended [40], with a 12-week monotherapy course prescribed for persistent replication beyond 3 months [41]. Although generally well tolerated, ribavirin can cause hemolytic anemia, particularly in patients with renal impairment due to its renal excretion [42]. It is contraindicated in individuals with a creatinine clearance below 50 mL/min and should be avoided in pregnant women due to its teratogenic effects [43].

Given the limited proportion of sustained responders, tailoring treatment to individual patients is essential for optimizing IFN-based therapy. For instance, in HBeAg-negative patients, the lack of HBsAg decline after 12 weeks of therapy predicts non-response, especially in genotype D patients [44]. Integrating baseline factors, like viral genotype, age, and ALT levels, with on-treatment predictors, such as HBV-RNA and HBsAg kinetics, could enhance personalized treatment and long-term outcomes.

2.2 Nucleoside/nucleotide analogues

NAs have become a cornerstone in the treatment of chronic HBV [15], and to a lesser extent, HCV [14]. Currently available NAs, include lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide, target viral polymerase to inhibit replication. They integrate into hepatitis viral DNA during replication, which causes premature chain termination and blocks the reverse transcription of RNA to DNA [45].

Lamivudine was the first nucleoside analogue to significantly inhibit viral replication, but its clinical utility was constrained by variable effectiveness and high resistance rates [46]. While about 40% of patients experienced HBeAg suppression after 3 years of treatment, resistance rates could reach 20% within the first year and escalate to 70% after 5 years, leading to viral breakthroughs and potential liver failure [45, 47]. Similarly, resistance with telbivudine can reach up to 22% in HBeAg-positive and 9% in HBeAg-negative patients [45, 48]. Moreover, lamivudine and telbivudine can cause mitochondrial toxicity, including myopathies and neuropathies [49], although lactic acidosis from lamivudine is rare [50].

Second-generation NAs, like entecavir, TDF, and tenofovir alafenamide, are highly potent antiviral agents with a strong resistance barrier, making them the preferred first-line treatments for chronic HBV [15]. These analogues inhibit HBV-DNA synthesis by competing with essential nucleotides, which reduces new hepatocyte infections, dilutes the intrahepatic cccDNA pool, and decreases its transcription. This cumulative effect gradually declines viral markers and modulates immune responses, potentially enabling some patients to control hepatitis infection without ongoing therapy. After around 5 years of treatment, 94–96% of HBeAg-positive and 95–99% of HBeAg-negative patients achieve undetectable HBV DNA levels [51]. TDF also demonstrates notable HBeAg loss in 49–53% of patients and a cumulative HBsAg loss of 11% [52].

Long-term NA treatment, well-tolerated oral medications, effectively suppresses viral replication over extended periods. Despite advancements, sustained therapy is often necessary due to the persistence of cccDNA, which can lead to viral reactivation after treatment discontinuation [53]. This extended use can result in resistance in up to 30% of patients, as incomplete viral suppression allows continued virion production and new hepatocyte infection [54]. Additionally, these treatments may cause nephrotoxicity and reduced bone mineral density [55]. Tenofovir alafenamide has emerged as a promising alternative, offering lower risks of renal and bone complications with improved potency and efficacy in normalizing ALT levels [56].

Recent strategies to overcome the limitations of monotherapies involve combining NAs with other agents, such as the virus entry inhibitor Myrcludex-B, which enhances antiviral activity by preventing intrahepatic viral spread and new infections [57]. This combination suggests that residual infective virions contribute to cccDNA persistence and transcription, as NAs do not impact cccDNA stability or transcription, leading to the ongoing production of viral proteins and pre-genomes [58]. Additionally, combining PEG-IFNα with entecavir has shown more promising outcomes than with lamivudine or telbivudine, with significant reductions in HBeAg and HBsAg levels. The combination of PEG-IFNα with entecavir or TDF may also offer more sustained suppression of viral markers, although efficacy may vary with hepatitis B virus genotypes, particularly for genotypes C and D [59].

NAs may be discontinued in HBeAg-positive chronic HBV patients without cirrhosis after achieving undetectable HBV-DNA and HBeAg seroconversion, followed by 6–12 months consolidation period [60]. Anti-hepatitis B e-antigen (Anti-Hb) seroconversion typically persists in over 85% of patients for at least 2 years post-discontinuation. However, HBsAg clearance is the preferred endpoint for discontinuation in HBeAg-negative patients, though clearance rates remain low. Serum viral DNA often reappears within 6 months after discontinuation, with relapse rates of 35% at 6 months and 55% at 2 years [61]. Notably, lower HBsAg levels at discontinuation correlate with higher rates of sustained virological response [60]. The current European Association for the Study of the Liver (EASL) guidelines recommend discontinuing NAs only in non-cirrhotic patients with at least 3 years of virological suppression, with close monitoring for potential relapse [15].

2.3 Direct-acting antiviral agents

The advent of DAA has revolutionized the treatment landscape for HCV, achieving SVR rates exceeding 97% with shorter treatment durations, around 8 to 12 weeks, and fewer side effects compared to previous regimens [62]. Contemporary oral DAA target essential viral non-structural proteins (NS) necessary for replication, specifically NS3/4A protease, NS5A, and NS5B polymerases. These antiviral agents are categorized based on their molecular targets, such as protease inhibitors (ending in “-previr”), NS5A inhibitors (ending in “-asvir”), and NS5B polymerase inhibitors (ending in “-buvir”). Despite these advances, about 4–5% of patients may not achieve viral eradication due to factors like poor adherence, relapse, advanced liver disease, or resistance mutations [63]. HCV’s rapid evolution can create resistance-associated variants, mainly in the NS5A region, but also in NS3 and NS5B [64].

Pangenotypic DAA combinations—such as sofosbuvir/velpatasvir (Epclusa), glecaprevir/pibrentasvir (Mavyret), sofosbuvir/ledipasvir (Harvoni), and sofosbuvir/velpatasvir/voxilaprevir (Vosevi)—are now the standard of care for treating HCV across all genotypes [14]. These regimens are effective even in patients with advanced liver disease and other comorbidities, with treatment choice guided by genotype, renal function, and concurrent medications [62]. However, protease inhibitors, primarily excreted through the liver, are contraindicated in patients with decompensated cirrhosis, those co-infected with human immunodeficiency viruses (HIV), or individuals who have undergone liver transplantation [65]. Additionally, sofosbuvir, a nucleoside NS5B inhibitor, is avoided in those with significant renal impairment [66].

Traditionally, treating acute HCV—defined as the first 6 months post-infection—was challenging and often required Peg-IFNα if viral replication persisted beyond 12 weeks. However, DAA now demonstrate effectiveness in the early stages of infection, particularly in high-risk populations, such as injection drug users, men who have sex with men, and individuals with HIV. In these groups, DAA can reduce morbidity, mortality, and transmission risk before the disease progresses to chronicity [67]. The EASL recommends an 8-week course of sofosbuvir/velpatasvir or glecaprevir/pibrentasvir for recently acquired HCV, although post-exposure prophylaxis with DAA is not advised without confirmed HCV transmission [14].

Liver transplantation remains a vital intervention for patients with HCV-related decompensated cirrhosis or HCC, despite a notable reduction in transplant demand due to the effectiveness of DAA [68]. Before the introduction of DAA, nearly all HCV-positive liver transplant recipients experienced recurrent infection post-transplantation, leading to severe complications such as fibrosis, recurrent cirrhosis, and fibrosing cholestatic hepatitis, with significantly reduced survival rates compared to HCV-negative recipients [69]. DAA have since dramatically enhanced the management of HCV in liver transplant candidates, improving overall survival and reducing graft rejection by preventing recurrence or achieving SVR when administered pre- or post-transplant [70]. This advancement extends to other solid organ transplants, making organ donation from HCV-positive donors more feasible and reducing waitlist mortality and healthcare costs [71]. HCV-negative recipients of HCV-positive organs are typically treated with DAA, either prophylactically or reactively upon detection of HCV RNA [72], with early studies showing high rates of SVR and graft survival outcomes [71].

While the simplicity of pangenotypic regimens marks a significant advancement, the high cost of DAA limits accessibility, particularly in low- and middle-income countries [73]. Ongoing concerns include resistance-associated variants, persistence of HCV RNA post-SVR, fibrosing cholestatic hepatitis, drug interactions, and challenges in treating co-infected or advanced liver disease patients. Some studies also report reduced efficacy of regimens like Vosevi in certain genotypes with cirrhosis [74]. Patients with advanced fibrosis remain at risk for liver complications and HCC, necessitating continued surveillance even after achieving SVR [75]. Although early fears suggested DAA could increase HCC recurrence by disrupting immune surveillance of pre-existing tumors, recent evidence refutes this risk, confirming SVR’s importance for improving liver function and long-term survival [76]. The timing of DAA initiation in HCV-positive HCC patients remains controversial, requiring adherence to updated guidelines to optimize outcomes and minimize risks [77].

3.1 Innovations in gene therapy

Gene therapy is emerging as a promising strategy, particularly for HBV. Central to this effort is the episomal cccDNA, an HBV replication intermediate that forms a minichromosome in the hepatocyte nucleus. A few copies of cccDNA can cause infection to rebound after treatment cessation through producing HBsAg in the bloodstream [83]. Current research focuses on gene therapies that silence cccDNA to achieve a functional or complete cure for HBV.

RNA interference (RNAi) offers a promising approach for treating chronic HBV by using small interfering RNA (siRNA) to target and suppress HBV mRNA selectively. A key advantage of siRNA therapies is their ability to target overlapping regions of the HBV genome, such as the X region, allowing a single siRNA to knock down all HBV transcripts [84]. However, efficient delivery to hepatocytes remains a challenge. This has been addressed by conjugating siRNA with ligands such as a triantennary N-acetyl galactosamine (GalNAc), which binds to the asialoglycoprotein receptor on hepatocytes and facilitates rapid endocytosis [85]. Early siRNA therapies encountered issues like off-target effects and hepatotoxicity. However, the modified siRNA VIR-2218, with improved stabilization, has shown a favorable safety profile and significant reductions in HBsAg levels, indicating its potential in future chronic HBV treatments [86].

Recent advancements emphasize the importance of co-delivery and optimized encapsulation systems. Researchers have developed a broad-spectrum siRNA encapsulated in a targeted lipid nanoparticle (tLNP) with 98.55% genotypic coverage. This system led to significant reductions in viral antigens and DNA levels in mouse models. Moreover, the co-delivery of siRNA with IL-2 mRNA further improved viral suppression, presenting a promising dual-modality therapeutic strategy [84]. Although RNAi therapies aim for a functional cure by reducing viral replication and many RNAi candidates have progressed to clinical trials, they often require repeated dosing and are used in combination with other treatments. This is where gene-editing technologies offer the potential to provide precise and permanent alterations to the viral genome that could eliminate its replication.

In addition to siRNA, other RNAi modalities, such as short hairpin RNA (shRNA) and gene-editing technologies like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have demonstrated potential in disrupting viral hepatitis DNA. For instance, lentiviral vectors have been used to express single, double, or triple shRNA cassettes targeting conserved viral sequences and host factors critical for HCV replication, without causing significant cytotoxicity [87]. ZFNs are engineered proteins combining DNA-binding abilities with nuclease activity for genome editing. By linking multiple Zinc-finger domains with a FokI nuclease, ZFNs can create site-specific double-strand breaks in DNA, enabling precise genome modifications [88]. Studies have shown ZFNs’ ability to disrupt HBV DNA and reduce viral replication, but most research has been limited to in vitro models, and concerns about off-target effects and cytotoxicity require further in vivo investigation [89].

Newer gene-editing technologies, like TALENs, have offered improved accuracy and safety profiles. Derived from Xanthomonas bacteria, TALENs function as dimers, utilizing the FokI nuclease for DNA cleavage. Compared to ZFNs, TALENs exhibit higher specificity, lower off-target effects, and reduced cytotoxicity, making them versatile tools against chronic viral infections like HBV [90]. In cell cultures and animal models, TALENs have effectively suppressed HBV replication by reducing HBsAg levels. By targeting both core and surface open reading frames, TALENs have shown robust antiviral effects, significantly reducing viral particles in vivo [91]. However, their efficacy is time-dependent, and results vary across different HBV genotypes and target sites. While TALENs have had some success in disrupting cccDNA, more research is needed to optimize their targeting of this critical viral reservoir.

The emergence of more precise gene-editing tools like CRISPR has overshadowed ZFN and TALEN technologies, although these earlier tools still offer a foundation for future antiviral strategies. The CRISPR/Cas system is a revolutionary third-generation gene-editing technology that uses guide RNA (gRNA) to direct the Cas protein to specific genomic sites, enhancing accuracy and efficiency [92]. In HBV research, CRISPR technology has shown promise by targeting regions of the viral genome with single guide RNAs (sgRNAs). Targeting the HBV X gene, for instance, has led to reductions in cccDNA, replication intermediates, HBsAg, and HBcAg [93]. In transgenic mouse models, CRISPR/Cas9, delivered via adeno-associated virus serotype 8 (AAV8), significantly reduced HBV core protein expression in the liver and lowered serum HBsAg levels, highlighting its antiviral potential [94].

Despite significant advancements in gene and RNA-based therapies, effective delivery of these synthetic nucleic acid effectors remains a significant challenge, and they face difficulty penetrating lipid membranes. The success of these therapies depends on developing advanced, functional delivery systems for in vivo use. Ongoing research is focused on next-generation delivery technologies and optimizing siRNA design to improve intracellular efficiency and extend therapeutic duration [95]. Continued innovation in delivery systems, combined with the refinement of therapeutic nucleic acids, offers great potential for overcoming delivery challenges, minimizing off-target effects, and advancing safer, more effective treatments—bringing us closer to functional cures for chronic diseases like hepatitis infection [12].

3.2 Targeted immunomodulatory therapies

In addition to gene therapies, immunomodulatory treatments are emerging as powerful tools for combating hepatitis infections. These therapies enhance the innate and adaptive immune responses, enabling the body to better fight the virus. By boosting immune activity or restoring exhausted immune cells, immunomodulatory therapies can complement gene therapies, offering a multifaceted approach to managing or potentially curing chronic hepatitis infections. Toll-like receptors (TLRs) are pivotal in the innate immune defense, recognizing pathogen-associated molecular patterns (PAMPs) and initiating cytokine production through various signaling pathways. Specifically, TLR-7 and TLR-8 agonists help induce IFN production, activate IFN-stimulated genes (ISGs), and trigger signaling cascades like JAK/STAT. The TLR-7 agonist GS-9620 has demonstrated the ability to suppress HBV through type I IFN induction in human hepatocyte cell lines, though it does not reduce cccDNA [96]. However, when combined with the TLR-7 agonist RO7020531 with a capsid assembly modulator, HBV DNA and HBsAg levels were significantly reduced in a mouse model [97]. In healthy human volunteers, TLR-7 agonists induced IFN-alpha and ISG expression, though further studies are needed to evaluate their efficacy in combination with other antiviral agents [98].

Recent advancements in chronic HBV therapies have focused on enhancing innate immunity. Retinoic acid-inducible gene-I (RIG-I) agonists, such as Inarigivir (SB 9200), activate immune pathways, leading to reduced HBV DNA and RNA levels. Clinical trials have shown that 22% of patients experienced HBsAg reduction, and higher doses of Inarigivir combined with tenofovir are currently under investigation [99]. Additionally, checkpoint inhibitors, such as the PD-1 inhibitor nivolumab, aim to restore T-cell function. Trials have demonstrated significant HBsAg reduction in some patients, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) blockade has also shown potential in improving immune responses against HBV [100]. These therapies represent promising steps toward more effective treatment strategies for chronic HBV.

Among the most innovative strategies for treating chronic HBV are T-cell-based therapies. In chronic HBV patients, T-cell exhaustion often impairs immune function, hindering effective viral control [101]. One promising approach involves generating functional HBV-specific T cells from patients and re-infusing them to overcome immune energy. Evidence from bone marrow and organ transplant cases suggests that adoptive transfer of engineered antigen-specific T cells can control HBV. These T cells, engineered via viral vectors to recognize HBV antigens, have shown encouraging results in HBV transgenic mice [102]. However, challenges such as the large-scale production of engineered T cells and complex regulatory and technological hurdles remain barriers to their widespread clinical application.

3.3 Advances in therapeutic vaccination

Therapeutic vaccines for chronic HBV have been explored to modulate the adaptive immune system and overcome immune exhaustion. In early attempts, an antigen-antibody complex vaccine (HBsAg-HBIG) with alum as an adjuvant (YIC) was tested to overcome immune tolerance. While the YIC vaccine initially showed promise, overstimulation led to immune fatigue, reducing its efficacy and underscoring the need to optimize immunization strategies [103]. Recent approaches, like ABX-203 (HeberNasvac), a vaccine containing HBsAg and HBcAg, have shown promise in clinical trials, achieving better viral load reduction and HBeAg seroconversion compared to Peg-IFNα [104]. Vector-based vaccines, like GS-4774 and TG-1050, which use viral vectors to encode HBV antigens and enhance T-cell activation, have demonstrated strong immune responses. However, these vaccines have yet to significantly reduce HBsAg levels in treated patients [105, 106]. Breaking immune tolerance remains a critical challenge in chronic HBV infection. For example, TG-1050 has demonstrated safety and immunogenicity, but pre-existing adenoviral immunity can limit its effectiveness, even though it has stimulated anti-HBV immune responses and activated cytotoxic functions in animal models [106]. Despite their limitations, combining these vaccines with antiviral agents or improving delivery methods may boost their therapeutic potential for HBV treatment.

Recent advancements in HCV vaccine development are focused on overcoming past challenges, such as the virus’s high genetic variability, lack of suitable animal models, and limited understanding of protective immune responses. Approaches like mRNA vaccines offer rapid testing of potential candidates and aim to stimulate strong, multi-specific cellular immune responses, including helper and cytotoxic T cells, along with high-titer, long-lasting, cross-reactive anti-envelope antibodies. HCV envelope glycoproteins, particularly E1 and E2, have become promising vaccine targets [107]. New approaches to vaccine design emphasize the identification of neutralizing antibodies associated with viral clearance by creating epitopes, like E2, to stimulate potent antiviral antibodies, such as HC33.1 and AP33, which have shown broad and robust HCV neutralization in experimental models [108].

Another promising strategy uses dendritic cells (DC) expressing HCV-derived core or NS3 antigens to activate autologous T cells, leading to cytokine production and strong immune responses [109]. Additionally, GS-9620, a small molecule agonist targeting DC receptor toll-like receptor 7 (TLR7), was developed to activate DCs, functioning as innate immune-like vaccines [96]. Although GS-9620 did not significantly reduce HBsAg levels in clinical trials, it enhanced HBV-specific T cells and NK cell responses in patients receiving NUC treatment [110]. Another example, GI-13020 (GS-4774), a heat-killed recombinant yeast expressing an HBx-HBs-HBc chimeric protein, elicited both CD4+ and CD8+ T-cell responses with acceptable initial tolerability. However, in Phase 2 trials, it did not lead to significant reductions in HBsAg levels [111].

A recent study highlights the vital role of adjuvants in boosting vaccine efficacy by enhancing innate immunity. Researchers examined gene expression in naive individuals vaccinated with hepatitis B surface antigen alongside various adjuvants, identifying a core innate immune signature that emerged after the second dose. This signature included the positive regulation of INF-related responses and activation of innate immune cells, which correlated with stronger antibody production. Notably, the adjuvant AS01B was especially effective in generating this inflammatory signature after the first dose, indicating that its success may depend more on its ability to elicit consistent immune responses across individuals rather than the specific receptors or pathways it targets [112].

While therapeutic vaccines hold great promise in strengthening immune response against chronic hepatitis infections, several challenges remain. One significant limitation is the variability in individual immune responses, which can affect the overall efficacy of the vaccine [113]. Genetic background, pre-existing immunity, and co-infections can influence patient responses to vaccination [114]. Additionally, the risk of adverse effects, including autoimmune reactions, poses a concern that requires careful consideration in vaccine design and implementation [115]. Addressing these challenges is essential for successfully integrating therapeutic vaccines into treatment protocols for chronic hepatitis infections.

4.1 Advancements in next-generation gene sequencing

Next-generation sequencing (NGS) has emerged as a transformative tool in clinical virology by providing significantly enhanced sensitivity compared to traditional Sanger sequencing, particularly in detecting drug resistance mutations, mixed genotypes, and viral quasi-species, including minor circulating variants [120]. Second-generation sequencing techniques have proven particularly adept at identifying and quantifying viral mutations and recombination events [121], while third-generation sequencing enables full-genome analysis [122]. NGS has also been pivotal in discovering novel viruses, including arenaviruses, Zika, and Ebola [123, 124, 125]. Additionally, it has facilitated the characterization of viral populations in animal and human hosts within natural environments [126, 127].

In hepatitis, NGS has significantly advanced the identification of HBV genotype mixtures, particularly in regions such as the preS and the 5′ end of the HBV X gene [128]. Despite its numerous advantages, a major limitation of NGS is the overwhelming amount of data produced, requiring advanced bioinformatic tools for proper interpretation [129]. While third-generation sequencing holds promise for comprehensive genome exploration, its clinical applicability remains hindered by higher sequencing error rates [122]. Additionally, there are challenges in variant interpretation across diverse clinical settings, which can complicate hepatitis diagnosis and prognosis [130].

4.2 Precision pharmacogenomics

Pharmacogenomics has revolutionized the personalization of drug prescriptions by exploring how individual genetic variations influence drug response, focusing primarily on pharmacokinetics and pharmacodynamics [131]. The main goal is to optimize therapeutic efficacy and minimize adverse drug reactions by tailoring treatments and risk assessments to an individual’s genetic profile [132, 133]. This field incorporates data from the Human Genome Project and other genetic databases to advance genomic-based therapies and drug discovery [134]. Genome-wide association studies (GWAS) remain the gold standard for identifying pharmacogenomic associations, despite the high cost and labor intensity associated with these studies [135, 136, 137].

GWAS primarily focus on identifying single nucleotide polymorphisms (SNPs) that influence the development and progression of liver diseases, including hepatitis and hereditary liver disorders. These SNPs contribute to differences in immune responses among HBV-infected patients, which can range from asymptomatic infections to severe conditions such as cirrhosis and end-stage HCC [138]. Several studies have identified polymorphisms in the HLA complex, which regulates innate and adaptive immunity during infections, as being associated with varying clinical outcomes in chronic HBV and HCV infections, influencing both viral clearance and progression to chronic stages [139]. Additionally, various HLA polymorphisms linked to the response to the HBV vaccine and infection persistence have been highlighted by several studies [140, 141]. Notably, SNPs within the HLA-DPA1 (rs3077), HLA-DPB1 (rs9277535), and HLA-DQ (rs2856718 and rs7453920) have been linked to chronic HBV outcomes, especially in Asian and American populations [135, 142, 143]. Protective HLA alleles, such as DQB106:03:01 and DRB113:01:01, have also been identified in Romanian populations, underscoring the geographical and ethnic variability in genetic susceptibility to HBV [144]. Beyond HLA polymorphisms, other genetic variants, such as those in ZNF208, have been implicated in HBV pathogenesis [145].

Host genetic factors, including IL28B, interferon-λ3, interferon-λ4, IL-12, IL-10, and TLRs, are strongly associated with treatment outcomes and disease progression [146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161]. The IL28B genotype has been a significant predictor of SVR in HCV patients undergoing treatment with Peg-IFNα and ribavirin. Variants, such as rs12979860, have shown a strong association with treatment success, particularly in Caucasian individuals, where those with the CC genotype achieve SVR at a rate six times higher than those with the CT or TT genotypes [162, 163, 164, 165]. Furthermore, genetic variants in TLRs, such as rs3775290 “CC” genotype, have been linked to the severity of liver fibrosis, cirrhosis risk, and HCV treatment outcomes by modulating immune responses through inflammatory cytokine production [166, 167]. The role of genetic polymorphisms in interferon lambda (IFNL) genes in viral clearance and liver disease progression further supports the importance of genetic profiling in managing HBV and HCV infections [168]. Interferon regulatory factors (IRFs), essential transcription factors in immune cell development, have also been associated with protection against HCV infection and cirrhosis by reducing the frequency of the IRF3 “AG” genotype [169, 170]. Additionally, emerging evidence directly links microRNAs (miRNAs) with HCV infection and HCC development, emphasizing the importance of investigating their regulatory roles in disease progression [171, 172].

Despite the promise of pharmacogenomics, the field encounters several challenges, primarily the high costs of large-scale GWAS and the substantial expenses related to data storage and analysis. These financial burdens also raise ethical concerns, especially around patient privacy and potential impacts on insurance coverage [173]. Additionally, genetic findings often vary across ethnic populations, and the absence of standardized workflows for variant interpretation further limits the universal applicability of pharmacogenomic insights in clinical practice [174]. Although debates persist on the impact of genetic polymorphisms in HBV outcomes, the genetic findings are anticipated to improve individualized risk assessments and vaccination approaches, aiding in the effort to eliminate hepatitis infections as a global health threat by 2030 [12].

4.3 Integration of mass spectrometry

Mass spectrometry (MS) has become an indispensable tool in virology, enabling detailed analysis of viral capsid proteins, viral mutants, post-translational modifications, and intact viruses [175, 176], advancing antiviral drug development and evaluation [177]. Compared to NGS, MS offers a cost-effective, user-friendly alternative and has been successfully applied to genetic typing, the detection of viral biomarkers, and advancing antiviral drug development and evaluation [178]. MS applications in virology have evolved two main paths: the development of specialized techniques for measuring viral physicochemical properties and the adaptation of proteomics and interactomics approaches to address virology-specific challenges.

MS techniques such as charge-detection MS (CDMS), ion mobility spectrometry (IMS), and gas-phase electrophoretic mobility molecular analysis (GEMMA) have proven valuable in analyzing high molecular weight molecules, including viruses [179]. Adaptations of electrospray ionization-MS (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have demonstrated effectiveness in detecting HBV and HCV, identifying all eight HBV genotypes [179]. When combined with multiplex PCR, MALDI-MS has been used to analyze multiple human enteric viruses, including hepatitis E virus [180]. The MALDI-MS coupled with time-of-flight (TOF) analysis offers high sensitivity and throughput, facilitating the mass screening of HBV patients undergoing lamivudine treatment [181].

MS-based proteomics has also shown that alterations in the cellular proteome, such as fatty acid chain modifications, play a role in preventing the progression of chronic HBV infection to HCC [182, 183]. Additionally, LC-MS-based metabolomic profiling has enabled the identification of metabolic biomarkers that differentiate chronic HBV from liver cirrhosis and HCC, improving early diagnosis and personalized treatment approaches [184, 185]. For HCV, gas chromatography-MS (GC-MS) analysis has linked altered glucose metabolism and branched-chain amino acids to hepatocellular damage and hyperglycemia in HCV-infected patients [186]. Furthermore, GC-MS-based metabolomic studies have identified biomarkers predictive of HCC recurrence, combining glutamate with aspartate as well as glycerol with proline, offering new avenues for monitoring disease progression before and after treatment [187].

Despite its broad applications, MS faces limitations in sensitivity and specificity when applied to complex viral populations. The integration of MS into clinical practice requires further validation to establish standardized workflows for viral detection and analysis [173].