The HBV genome is a 3.2 kilobase relaxed circular, partially double-stranded DNA molecule. It has four partially overlapping open reading frames encoding the viral envelope (pre-S and S), nucleocapsid (precore and core), polymerase, and X proteins. After binding to hepatocytes, the virion is taken up into the cell by endocytosis and uncoated. The partially double-stranded DNA genome is converted to a covalently closed circular DNA (cccDNA) in the cell nucleus. The cccDNA is used as a template for transcription of the pregenomic RNA (pgRNA) and messenger RNA in the cell nucleus. The pgRNA moves into the host cell cytoplasm and serves as the template for translation of the HBV reverse transcriptase (RT) as well as the core protein by the cellular translational proteins. Concurrently, the HBV RT reverse transcribes the pgRNA to a new circular DNA molecule. Early in the replication cycle, some of the newly synthesized genomes will circulate back to the nucleus to maintain and increase the pool of cccDNA [3].

Although HBV is a DNA virus, it replicates by a RT that lacks proofreading activity and, as a result, is prone to errors. The overlapping open reading frames of the genome limit the types of mutations that can be tolerated. However, variations in HBV sequences have been detected in almost all regions of the genome. Consequently, HBV exists as quasispecies, and different patients may be infected with different strains and genotypes.

Seven phylogenetic genotypes (A through H) of HBV have been identified, most of which have distinct geographic distribution. Genotypes are defined by intergroup divergence of greater than 8 % in the complete genome nucleotide sequence. All known genotypes have been found in the USA with the prevalence of A, B, C, D, and E–G being 35 %, 22 %, 31 %, 10 %, and 2 %, respectively [4]. Recent data suggest that HBV genotype plays an important role in the progression of HBV-related liver disease as well as response to interferon alpha (IFN-α) and pegylated IFN-α; however, HBV genotyping is not necessary in routine clinical practice [5].

Serologic assays with high levels of sensitivity, specificity, and reproducibility have been developed to detect HBV antigens and their respective antibodies. This complicated system of serologic markers is used for diagnosis of HBV infection and to define the phase of infection, degree of infectivity, prognosis, and the patient’s immune status. The presence of HBV DNA in the serum is a marker of viral replication in the liver and has replaced hepatitis B e antigen (HBeAg) as the most sensitive marker of viral replication. HBeAg is the extracellular form of the hepatitis B core protein. Molecular assays to quantify blood levels HBV DNA are useful for the initial evaluation of HBV infections, monitoring of patients with chronic infections, and assessing the efficacy of antiviral treatment [3, 5]. In addition, US blood donors are routinely screened for HBV DNA by qualitative tests to detect donors in the early stage of infection [6]. Antiviral resistance mutations are detected by molecular methods that identify known mutations associated with drug resistance.

The initial evaluation of patients found to have HBsAg in serum should include routine liver tests and a variety of virologic tests including HBV DNA tests [5]. Chronic HBV infection is a disease of variable course, and establishing baseline laboratory values at the time of diagnosis is important clinically for the tracking of disease progression over time and to evaluate candidates for liver biopsy. Monitoring disease activity in chronically HBV-infected patients is best done by measuring aminotransferase (ALT) levels at regular intervals in HBeAg-positive patients. However, serial HBV DNA testing is recommended in HBeAg-negative patients. The determination of serum HBV DNA levels (viral load) is important in the pretreatment evaluation and monitoring of therapeutic response in patients with chronic infection [5]. Currently, therapy for chronic HBV infection does not eradicate the virus and has limited long-term efficacy. The decision to treat should be based on ALT elevations, the presence of HBeAg or HBV DNA viral load of >2,000 IU/ml or both, the presence of moderate disease activity and fibrosis on liver biopsy, and virologic testing to exclude concurrent infections with hepatitis D virus (HDV), HCV, and human immunodeficiency virus (HIV). The goals of treatment of chronic hepatitis B are to achieve sustained suppression of HBV replication and to prevent further progression of liver disease. Parameters used to indicate treatment response include normalization of serum ALT, decrease in serum HBV DNA level, and loss of HBeAg with or without detection of anti-HBeAg. Currently, eight US Federal Drug Administration (FDA)-approved therapies are available for treatment of chronic HBV infection: IFN-α, pegylated IFN-α2a, four nucleoside analogs (NS; lamivudine, telbivudine, entecavir, and emtricitabine) and two nucleotide analogs (NT; adefovir and tenofovir). Several factors predict a favorable response to IFN treatment with the most important being high ALT and low serum HBV DNA viral load, which are indirect markers of immune clearance.

Therapy usually does not eradicate the virus because of the difficulty of eliminating the covalently closed circular form of the HBV genome from the liver and the existence of extrahepatic reservoirs of HBV. Endpoints of treatment have traditionally been clearance of HBeAg, development of anti-HBe antibodies, and undetectable serum HBV DNA using insensitive hybridization assays with detection limits of approximately 10⁶ genome copies/ml. Achieving these endpoints usually is accompanied by resolution of liver disease as evidenced by normalization of the ALT level and decreased histologic activity on liver biopsy. The response usually is sustained at long-term follow-up. Nevertheless, most responders continue to have detectable HBV DNA when sensitive nucleic acid amplification tests are used. Responses to antiviral therapy are categorized as biochemical, virologic, or histologic and as on-therapy, or sustained off-therapy (Table 46.2) [5].

Several variations in the nucleotide sequence of HBV have important clinical consequences. An important mutation in the S gene is a glycine-to-arginine substitution at codon 145 (G145R) in the conserved “a” determinant, which causes decreased affinity of the HBsAg for anti-HBs antibodies [7]. HBV with this mutation has been found in children of HBsAg-positive mothers who develop HBV infection despite vaccination and an adequate anti-HBs antibody response after vaccination, as well as in liver transplant recipients who have recurrent infection despite administration of HBV immune globulin [8, 9]. These immune escape mutants have raised concern about vaccine efficacy and serologically silent infections. The G145R mutation has been reported in many countries and is responsible for 2–40 % of vaccine failures. Although there is diminished binding to anti-HBs antibodies, the vast majority of S mutants can be readily detected with the current generation of HBsAg tests. Thus, an initial concern that widespread use of HBV immune globulin and vaccination would result in HBV mutants that would escape detection in the HBsAg test was unfounded.

Mutations in the basal core promoter and the precore genes affect the synthesis of HBeAg and commonly arise under immune pressure [10]. The most common basal core promoter mutation has a dual change of A to T at nucleotide (nt) 1,762 (T1762) and G to A at nt 1,764 (A1764) that diminishes the amount of mRNA and hence HBeAg secretion [11]. The predominant precore mutation is a G to A change at nt 1,896 (A1896), which leads to premature termination of the precore protein at codon 28, thus preventing the production of HBeAg [12]. The A1896 mutation is infrequent in North America and Western Europe but is geographically widespread. This geographic variability in frequency is related to the predominant genotypes in a geographic region because the mutation is found only in genotypes B, C, D, and E.

The A1896 mutation was first reported in patients with chronic active hepatitis or fulminant hepatitis. However, the A1896 mutation also can be present in asymptomatic carriers and viruses with this mutation replicate no more efficiently than wild-type HBV. Thus, the pathophysiologic significance of this mutation is unclear [13]. However, the clinical picture of persistent HBV replication and active liver disease in HBeAg-negative patients appears to be increasingly prevalent, and in some regions the A1896-mutant virus may be more prevalent than wild-type virus.

Therapy for chronic hepatitis B requires long courses of treatment with NS or NT analogs. A major concern with long-term therapy is the development of antiviral resistance. The rate at which resistant mutants are selected is related to pretreatment serum HBV DNA viral load, rapidity of viral suppression, duration of treatment, and prior antiviral exposure. The incidence of genotypic resistance also varies with the sensitivity of the methods used to detect resistance mutations and the patient population tested. The definitions of terms used to describe resistance to NS and NT analogs are summarized in Table 46.3 [5].

Typically, when a patient experiences a virologic breakthrough, HBV resistance genotyping should be performed. The standardized nomenclature of HBV antiviral resistance mutations is shown Table 46.4 [3, 14, 15]. No HBV mutations are associated with resistance to IFN-α or pegylated IFN-α2a.

A major dilemma in the interpretation of serum HBV viral load results is what values should be used to define treatment indications and response. Because HBV DNA may persist in individuals who have serological recovery from acute infection, low levels may not be associated with progressive liver disease and viral clearance is an unrealistic treatment endpoint. An arbitrary value of 20,000 IU/ml (>10⁵ copies/ml) was chosen as a diagnostic criterion for chronic hepatitis B at the 2000 NIH conference [18]. However, progressive liver disease can be found in individuals with lower levels. In addition, wide fluctuations in HBV viral load from undetectable to >2 million IU/ml are observed in some patients with chronic hepatitis B [19, 20]. Consequently, serial monitoring is more important than reliance on a single cutoff value in determining prognosis and the need for treatment.

The first manifestation of antiviral resistance is an increase in HBV viral load >1 log₁₀ from nadir during treatment in a patient who had an initial virologic response. Virologic breakthroughs often are related to therapy noncompliance, so compliance should be assessed before testing for genotypic resistance. HBV viral load levels tend to be low initially because antiviral-resistant mutants have decreased replication fitness. However, compensatory mutations that can restore replication fitness often emerge during continued therapy leading to progressive increases in HBV viral load followed by biochemical breakthrough. Antiviral resistant mutations can be detected months to years prior to biochemical breakthrough. Thus early detection of these mutations can prevent hepatitis flares and liver decompensation. Cross-resistance mutations do occur that limit future treatment options and multidrug-resistant mutants in patients who have received sequential monotherapies have been described [21].

A World Health Organization (WHO) international HBV standard was created in 2001 in response to the recognized need to standardize HBV DNA quantification assays [22]. The WHO HBV standard is a high-titer, genotype A virus preparation (code 97/746) which was assigned a potency of 10⁶ IU/ml. A second standard (code 97/750) was established in 2006 and is quantitatively equivalent to the first standard. The standard established that 1 IU was equivalent to 5.4 genome equivalents by testing the pooled material with a range of commercially available and laboratory-developed tests. However, despite the availability of HBV DNA standards, the various quantitative assays usually have different conversion factors for copies to IU/ml, which may reflect their different amplification and detection chemistries. Laboratories should report HBV viral load test results in IU/ml as both log₁₀ transformed and arithmetic values.

As with any viral load test, some of the variability in quantification may be attributable to sample processing. Either serum or plasma can be used for most tests. Whole blood should be centrifuged within 6 h of collection and the serum or plasma removed from the cells. Short-term storage of serum or plasma is safe at refrigerator temperatures (4 °C). Freezing at temperatures ≤20 °C is recommended for long-term storage of samples.

The standardized nomenclature for reporting of HBV antiviral resistance mutations shown Table 46.4 should be used when resistance genotyping is performed. The inability of genotyping assays to detect minor populations of circulating HBV is a significant technical issue. In general, direct sequencing is limited to resolution of populations which are ≥20 % of the viral population. HBV is included in the hepatitis viral load proficiency testing surveys available from the College of American Pathologists.

Molecular assays for quantification and genotyping of HBV will become more fully integrated in the testing strategies for the diagnosis and management of patients with chronic hepatitis B. Clinical practice guidelines now reflect the importance of HBV viral load and genotyping tests in improving patient outcomes and this is likely to create more demand for these tests. The current generation of real-time PCR viral load assays meets the clinical needs and development and adoption of the WHO international standard has improved the agreement between results of different assays. Improved HBV treatment will depend on development of better laboratory tools for assessing genotypic resistance. Genotyping beyond individual mutation detection to a more comprehensive genomic analysis to predict fitness and other polymutational phenotypes, and detection of minor resistant populations will be possible with next-generation sequencing methods [22, 24].

HCV is an RNA virus with a positive-sense, single-stranded genome of approximately 9,500 nt encoding a single polyprotein of about 3,000 amino acids. The long open reading frame is flanked at each end by short untranslated regions (UTR). The genome structure is most similar to viruses of the family Flaviviridae, which includes many of the arthropod-borne viruses. As in other flaviviruses, the three N-terminal proteins of HCV (core, envelope 1 [E1], and envelope 2 [E2]) are probably structural and the four C-terminal proteins (nonstructural 2, 3, 4, and 5) are thought to function in viral replication. HCV is classified within the family Flaviviridae in its own genus, Hepacivirus.

The 5′ UTR is a highly conserved region of 341 nt and has a complex secondary structure. It contains an internal ribosome entry site and is important in the translation of the long open reading frame. The 3′ UTR contains a short region that varies in sequence and length, followed by a polypyrimidine stretch of variable length, and finally a highly conserved sequence of 98 nt, which constitutes the terminus of the genome. The function of the 3′ UTR is not known but is thought to be essential for viral replication.

The E1 and E2 regions of HCV are the most variable regions within the genome at both the nucleotide and amino acid levels. Two regions in E2, called hypervariable regions 1 and 2 (HVR1 and HRV2, respectively), show extreme sequence variability, which is thought to result from selective pressure by antiviral antibodies. E2 also contains the binding site for CD81, one of the putative cell receptors or coreceptors for HCV.

The nonstructural regions 2 (NS2) and 3 (NS3) contain a zinc-dependent autoprotease that cleaves the polyprotein at the NS2-NS3 junction. The aminoterminal portion of the NS3 protein also is a serine protease that cleaves the polyprotein at several sites. The carboxyterminal portion of the NS3 protein has helicase activity, which is important for HCV replication. The NS4A protein is a cofactor for NS3 serine protease. The NS5B region encodes the RNA-dependent RNA polymerase, which replicates the viral genome. A region in NS5A has been linked to IFN-α response and therefore is called the IFN-α-sensitivity determining region (ISDR).