Hepatitis B Virus, Genotypes and Subtypes

Abstract

Hepatitis simply means inflammation of liver. This word came from heap: the Latin for liver and “titis” means inflammation. In addition to viruses, many varieties of agents can cause hepatitis such as bacteria, parasites, fungi and chemical agents including drugs, toxins and alcohol. Hepatitis B virus is classified as an Orthohepadna virus (Genera) within the family Hepadnaviridae. This family Includes the wood chuck hepatitis virus WHV, the duck hepatitis virus DHBV, and several other avian and mammalian variants. The human HBV has been shown to infect chimpanzees, Barbary macaques and tree shrews. All hepadnaviridae have similar to hepatotropism and life cycles in their hosts. HBV infection is a global health problem which is 50–100 times more infectious than HIV. Approximately 400 million people are carriers of chronic liver disease every year due to consequences of the disease. Not only HBV can infect hepatocytes but also infects in extrahepatic sites including lymph nodes, bone marrow, circulating lymphocytes, spleen and pancreas. Hepatitis B virus can occur as an acute or chronic disease. Previously, HBV genotypes have been classified into eight genotypes (A-H) and because of genome diversity is a hallmark of HBV virus allowed its classification into (10) genotypes (A–J). The clinical relevance of such genotype is yet unclear. Detection of HBV genotype is very important to clarify the pathogenesis, rout of infection and virulence of the virus. The major classification of HBV subtype is sorted into 4 subtypes or serotypes (adr, adw, ayr, and ayw). The four possible combinations define the major subtypes and additional amino acids contribute to immunogenicity. These subtypes can be further classified into (9) serotypes (adw2, adw4q-, adrq+, adrq-, ayw1, ayw2, ayw3, ayw4 and ayr). Epidemiologic studies found that the prevalence of these serotypes varies in different parts of the world.

1. Introduction

Hepatitis simply means inflammation of liver. This word came from heap: the Latin for liver and “titis” means inflammation. In addition to viruses, many varieties of agents can cause hepatitis such as bacteria, parasites, fungi and chemical agents including drugs, toxins and alcohol [1, 2].

Currently, 11 types of viruses are recognize causing hepatitis, Epstein- Barr virus (EBV), Cytomegalovirus (CMV) and 9 of hepatotropic viruses. Only 3 out of these 9 viruses are well characterized from A-E. Hepatitis A (HAV) sometimes called infectious hepatitis. Hepatitis B (HBV) is called serum hepatitis. Hepatitis C (formerly none A or B hepatitis NABA). Hepatitis D (HDV) which is formerly enteric transmitted hepatitis. Newly discovered forms of viral hepatitis including hepatitis F (HFV), hepatitis G (HGV), Transfusion Transmitted virus (TTV) and SEN virus. They all predominantly affect and infect liver cells. Despite significant overlap in the clinical manifestation caused by them, these types of viruses differ widely in their morphology, genomic organization, taxonomic classification and mode of replication [2, 3, 4].

Hepatitis B infection is a global health problem which is 50–100 times more infectious than HIV. Approximately 400 million people are carriers of chronic liver disease every year due to consequences of the disease [5, 6, 7]. Not only HBV can infect hepatocytes but also infects in extrahepatic sites including lymph nodes, bone marrow, circulating lymphocytes, spleen and pancreas. Hepatitis B virus can occur as an acute or chronic disease [8].

People at high risk of infection including those requiring frequent transfusions or hemodialysis, physicians, dentists, nurses, and other health care workers, intravenous drug users, police, firemen and others who are likely to come into contact with potentially infected blood products [9], as well as, sexual contacts with an acute or chronically infected persons. In the US, homosexually active men consist of 6%, whereas heterosexually with multiple partners consist of 0.5% from all risk factors [10].

Approximately 5% of the infected world’s population may lead to cirrhosis and HCC worldwide. It is approximated that (500, 000 to 1000, 000) persons die annually from HBV related liver disease. Most infections occur at birth or during early childhood. Infections usually cluster in households of chronically infected patients [11].

2. Acute hepatitis B

Acute disease typically occurs in the infected adolescents or adult who have not been vaccinated. This acute presentation can be life threatening due to massive liver damage from the host immune reactions [12]. Most people with HBV experience few or no symptoms; in fact, a 65% are unaware that they carry the virus. Although, a 30% of people with acute hepatitis B have no symptoms and most people with chronic HBV also have few or no symptoms, most symptoms may include fatigue (unusual, prolonged tiredness), fever, malaise (a flu-like feeling), nausea, vomiting, yellowing of skin and eyes (Jaundice), loss of appetite (anorexia), abdominal pain or bloating, indigestion, headache, itching (pruritus) and muscles or joints aches [13]. Acute hepatitis may in some cases progress to fulminant hepatitis leading to liver failure, which is a state with high mortality [6]. The weak immune response generated by young children acutely infected hepatocytes. For this reason, clinical symptoms suggestive of acute HBV infection are frequently absent in this patients population. For those patients who resolve their infection, HBsAg disappears at about 3–6 month, often just prior to the detection of antibodies to hepatitis B surface antigen (anti-HBs), while some patients with self-limited infection, however may still have low levels of HBV DNA in blood; whether the HBV DNA is a part of intact virions remains unknown [14].

In some people, the hepatitis B virus can also cause a chronic liver infection that can later develop into cirrhosis (a scarring of the liver) or liver cancer (http://virology-online.com/viruses/HepatitisB.htm) (Figure 1).

Figure 1.

Acute hepatitis B virus infection with recovery typical serological course. http://virology-online.com/viruses/index.htm.

3. Chronic hepatitis B

Chronic HBV (CHB) infection can be define as the presence of hepatitis B surface antigen (HBsAg) in the serum of an infected individuals for at least six months or as the presence of HBsAg in a patient who is negative for immunoglobulin M antibodies to the hepatitis B core antigen (anti-HBc).

Chronic hepatitis can be divided into four stages. The first stage, the immune tolerance phase which is characterized by active viral replication and immune system tolerance. In this initial phase, HBV DNA replicates at a high levels and the HBsAg and HBeAg can be detectable while the Aminotransferase (ALT) levels are normal or low, mild or no liver necroinflamation and no or slow progression of fibrosis. In this phase, more prolonged in subjects infected prenatally or in the first years of life. Next, the immune clearance phase: The immunologic response is causing inflammation and hepatic injury as a result of viral clearance. Here, the ALT levels are elevated and moderate/severe necroinflammation in liver biopsy is observed. The third phase, inactive carrier state: The viral clearance is accompanied by seroconversion of HBsAg, resulting in relatively low HBV DNA level and normalized ALT levels. Few patients reach the final stage, when the HBsAg is completely cleared and anti-HBs becomes detectable as a sign of immunity [6, 13, 15]. The risk of developing chronic hepatitis B infection that depends on the age at which infection is acquired. The risk is the lowest in adults and > 90% in neonates whose mothers are HBeAg-positive. Chronic infection is less frequent in those infected as the children. The risk of becoming chronically infected with hepatitis B is increased in those whose immunity is impaired [16]. Clinically, the e-antigen HBeAg is important in chronic infection as it is regarded a marker for replication and indicative of ongoing infection. When seroconversion occurs, it normally reflects remission of liver disease and viral clearance [6]. Persons with chronic HBV may have HBeAg or anti-HBe in their sera. In persons who are HBeAg positive, spontaneous seroconversion from HBeAg to anti-HBe commonly occurs with often accompanied by a flare in aminotransferase ALT levels. After conversion of HBeAg to anti-HBe, most persons have normal ALT levels and lower levels of HBV DNA which is usually <10³ copies / ml (Figure 2) [17].

Figure 2.

Progression of chronic hepatitis B virus infection typical serological course. http://virology-online.com/viruses/index.htm.

4. Occult hepatitis B infection (OBI)

In this stage of infection, HBV DNA in the serum or in the liver may in some cases still be detectable in the absence of HBsAg which is termed occult hepatitis B. The clinical importance of this is not completely understood, but occult hepatitis B has been associated with reactivation in the setting of immunosuppression and enhancing risk for liver cancer [6, 18]. This type of infection represents a potential transmission source of HBV via blood transfusion or organ transplantation. In addition, occult HBV infection has been associated with cryptogenic CH and HCC. Furthermore, some studies suggested that occult hepatitis B might affect responsiveness of chronic HCV to interferon therapy and disease progress [9].

5. Cirrhosis and hepatocellular carcinoma (HCC)

Some reports have been estimated that up to 40% of individuals with chronic hepatitis B (CHB) will progress to cirrhosis [13, 19, 20] and may lead to hepatocellular carcinoma (HCC). Worldwide, more than 50% of HCC cases, and in highly endemic areas 70–80% of HCC cases are attributable to HBV and 20% of the 400 million people with chronic hepatitis B infection will develop to HCC. It has been showed that the presence of HBeAg and higher levels of HBV DNA have been found to be strong risk factors for HCC in patients with chronic HBV infection and mainly develops in patients with liver cirrhosis [6, 19, 21]. The mechanisms of oncogenesis by HBV remain obscure. HBV may stimulate active regeneration and cirrhosis which may be associated with long-term chronicity. However, HBV associates tumors occasionally arise in the absence of cirrhosis, and such hypotheses do not explain the frequent finding of integrated viral DNA in tumors. Although insertional mutagenesis of HBV remains an attractive hypothesis to explain its oncogenicity. Like many other cancers, there is insufficient supportive evidence development of hepatocellular carcinoma likely to be a multifactorial process [22]. The incidence of HCC may also be affected by factors other than HBV infection such as HCV co-infection, alcohol intake and aflatoxin B1 in the food supply. In the Amazonian basin, the genotype F infections are associated with fulminant hepatitis, but this occurs in the context of co-infection or super infection with Hepatitis Delta Virus (HDV) genotype III [17, 23].

Many other risk factors have been implicated in the progression of liver disease and the development of HCC. In such co-infections have been reported that HBV may carriers with more than one genotype. Some common co- infection occurs between genotype B and C, A and D, which is presumably due to the coexistence of these genotypes in the same regions. Recombination between genotypes has been reported as genotypes A with D [24]. The clinical impact of co-infections is unclear, but the viral loads have been reported to be higher in the co-infected patients. The frequency of co- infection may be associated with genotyping method as the reported frequency varies widely [6]. Persons who are co-infected with both HBV and HCV also have an increased risk of developing HCC, as compared with those who are infected with either virus alone. Even though, co-infection with HDV has not been shown to increase the risk of HCC. One study demonstrated that HCC appears at younger ages in co-infected persons than it does in those infected solely with HBV. Using chronic alcohol also appears to increase the risk of cirrhosis (Figures 3 and 4) [17].

Figure 3.

EM of HBV particles, https://www.google.com/search?source=univ&tbm=isch&q=hepatitis+B+virus+electron+microscope+images&client=opera&sa=X&ved=2ahUKEwiTsoLLsajwAhVSNOwKHXCvCuoQjJkEegQIAxAB&biw=1366&bih=658.

Figure 4.

The architecture of a Dane particle. https://people.rit.edu/japfaa/infectious.html.

6. HBV genotypes

The clinical relevance of such genotype is yet unclear. However, because the HBV induced disease is the resultant of virus-host interaction, the disease characteristics may be influenced by the genotypes of the virus [5]. HBV genotype and subgenotype are strong factors in predicting outcomes of chronic HBV infections [25].

Traditionally, HBV genotypes has been based on one of the following criteria: an intergroup divergence of 8% (similarity in 92%) [26, 27] or greater over the complete genome sequence, or 4 ± 1% or greater divergence of the surface antigen HBsAg [28]. Detection of HBV genotype is very important to clarify the pathogenesis, rout of infection and virulence of the virus [29]. In the context of the findings described, there might be a need to further differentiate between genetic variants versus genotypes [30]. Since the HBV genotype is due to the entire nucleotide sequence, the HBV genotype is more appropriate for investigation of geographic distribution and epidemiologic connections [31]. Previously, HBV genotypes have been classified into eight genotypes (A-H) and because of genome diversity is a hallmark of HBV virus allowed its classification into (10) genotypes (A–J) [32, 33]. Genotypes A-D were identified in 1988 under the sequence divergence in the entire genome exceeding 8%, and designated by capital letters of the alphabet [34]. Genotype E-F were identified in 1993 and genotype G was identified in 2000. Genotype H which is phylogenetically closely related to genotype F was proposed in 2002 [6]. Genotype I has been described and isolated from Hanoi in the northern part of Vietnam, Laos, a primitive tribe from northeast India as well as in the northwest of China [18, 35, 36]. Finally, the newest genotype J was identified in the Ryukyu Islands in Japan and this genotype has a close relationship with gibbon/orangutan genotypes, and human genotype C [37].

Zekri and coworkers found that HBV mixed genotype infections could probably be of clinical significance in HBV-induced liver diseases. He established that prevalence of mixed A/D genotype infections related to induce chronic liver diseases and evaluation of therapy [38].

6.1 Relationship between HBV genotypes

There are structural, functional, infectivity and clinical differences between HBV genotypes. Such differences include prognosis, progression of disease, complications as cirrhosis and hepatocellular carcinoma, as well as response to antiviral therapy. Structurally, HBV genotypes differ in the length of their genomes. The numbering of HBV genome from the EcoRI site leads to difficulties in comparing nucleotide positions between genotypes. Functionally, the Pre-S region that is important for virus attachment and cell entry, shows momentous differences between genotypes. Genotype A differs mainly in sequence of the Pre-S2 region, and has insertion of six nucleotides in the terminal protein portion of the polymerase gene overlapping the core gene, and shares some structural features with genotype F. Genotype D genome is most similar to Genotype E, especially in the X-gene. Differences in RNA splicing folding between genotypes could be predicted [39].

A recent study by Chan et al. indicated that genotype C was associated with more severe liver fibrosis than genotype B probably because of delayed HBeAg seroconversion and prolonged active disease [20, 40]. The major structural differences between HBV genotypes are shown in Table 1 (2.1) below depending on nucleotide numbering, length and characteristic indels of HBV genotypes:

Genotype	Length	Insertion/Depletion
A	3221	Insertion core: 6 bp
B	3215
C	3215
D	3182	Depletion Pre-S1: 33 bp
E	3212	Depletion Pre-S1: 3 bp
F	3215
G	3248	Insertion core: 36 bp, Depletion Pre-S1: 3 bp
H	3215

Table 1.

Differences between the main HBV genotypes [6].

7. Prevalence and epidemiology of genotypes and subgenotypes

Humans are only reservoir for HBV, which is 50–100 times more infectious than HIV. The prevalence of HBV infection varies in different parts of the world, with most of the disease burden occurring in Asia and Africa [41].

8. Genotype A

Genotype A derived mainly from Europe, India, Africa, and North America [42]. The existence of subgenotypes within genotype A has been reported (A1/Aa) from South Africa and South Asia. Subgenotype Ae/A2 is mainly endemic in Europe and United States. Ac/A3 is mostly found among populations of West and Central Africa [21]. These subgenotypes were significantly distinguished by bootstrap at phylogenetic analyses complete genomes. The differences between European and Afro-Asian of genotype A strains that the subgenotype A1 strains encoding Asn (207) and Leu (209), while the A2 strains had Ser (207) and Val (209). All strains specifying ayw1 serotype belonged to A1, and most of them were from Africa. Genotype A is corresponding to subtype adw [8, 42].

8.1 Genotype A and its subgenotypes

Genotype A is distinguished at the carboxylic end of the core gene by a 6 nucleotide insert. A comprehensive analytical study of genotype A led to classification of A1, A2, A4, and A3 subgenotype, as the latter sequence category did not follow the subgenotype classification criterion. The subgenotypes A1, A4 and A3 are mostly present in Africa, while A2 prevails in Northern, Central and Northern Europe and in North America [8, 42].

9. Genotype B

Genotype B is originated mainly from China, Japan, and Southeast Asia (Vietnam, Thailand, and Indonesia). Four subgenotypes, designated B1–B4, were confirmed by significant bootstrap when comparing complete genomes [24, 42]. Other classification of genotype B isolates into two groups: Bj (“j” stands for Japan), mostly found in Japan, and Ba (“a” stands for Asia) [43]. All strains specified adw2 serotype with the exception of the strains in B4 which specified ayw1 or adw3 serotype according to the strain. Subgenotype B1 was formed mainly by 18 of the 25 S genes of genotype B strains from Japan, corresponding to the described group Bj while the most genotype B strains from China belonged to subgenotype B2 which also comprised strains from Vietnam. Subgenotype B3 was formed by four strains from Indonesia. Subgenotype B4 comprised only strains from Vietnam. Apart from the Arg (122) in B4, there were no amino acid substitutions in HBsAg characteristic of individual subgenotypes within B [42].

9.1 Novel subgenotypes of genotype B

The subgenotypes of B have been reclassified into six subgenotypes, namely: B1, B2, B4–B6 and quasi-subgenotype B3 according to the new classification with a phylogenetic and sequence divergence of >4 percent. In contrast to the remaining subgenotypes of B that have this mixture, subgenotype B1 (previously Bj), found in mainly Japan and B5 (previously B6) from the Canadian Inuit population, are genotype B without recombination with Genotype C in the precore/core area. Subgene B1 was presumably B5’s ancestor, likely transported from Siberia and Alaska to North America and Greenland by indigenous peoples during the migration [42].

10. Genotype C

Genotype C genome shows four subgenotypes, C1–C4 [21] supported by 96–100% bootstrap with clear geographical clustering. The ayr subtype is widespread in genotype C. Australian strains specified ayw3. In the subgenotypes C1–C3, there were an intermixture within the adr strains of strains specifying adw2 or ayr. The constraints against substitutions of subtype specifying residues122 and 160 thus seem less pronounced for genotype C than for the other genotypes. C1 was formed by the majority of the strains from the Far East (Japan, Korea, and China) [42]. Sakamoto found a novel subgenotypes of HBV/C5 and HBV/B5 among chronic liver disease patients in the Philippines [44].

10.1 Novel subgenotypes of genotype C

Genetic C is the earliest HBV genotype, according to Paraskevis et al. The C1–C16, which represents a longer period of endemicity in humans, is the largest number of subgenotypes. In Indonesia, there are a significant number of sub-genotypes. Subgenotype C4 is solely present in northern Australian aboriginal people that came down from a group of establishments who emigrated at least 50.000 years ago from Africa [44].

10.2 Genotype D

Genotype D is the most widespread genotype and predominated in the Mediterranean area, the Near East, and as far as India. It was also found in Aboriginal populations in Asia from Indonesia and Papua. The strains specified ayw2, ayw3, or ayw4 serotype with the exception of two European strain specifying Lys (122), Thr (127), and Lys (160) corresponding to the putative subtype adw3. Phylogenetic analysis of complete genomes have been distinguishing four subgenotypes D1–D4. Strains specifying ayw2 were found in D1, D3, and D4. The geographical distribution of the subgenotypes within D was less restricted than that of genotypes A, B, and C. Although, the strains from Middle East mainly belonged to D1 [25] those from South Africa and Alaska mainly to D3, while those from Oceania and Somalia were only found in D4 [42]. Genotype D is currently segregated into eight subgenotypes (D1–D8). A novel D9 isolates do not possess any unique motif in the Pre-S/S ORF that can distinguish them from the other eight subgenotypes of D. D9 subgenotype is originated from discrete recombination events between genotypes D and C as evident from the fact that both genotype C and D9 sequences are monophyletic in the core region [32]. Genotype D is characterized by a 33 nucleotide deletion at the N-terminus of the Pre-S1 region, therefore it has the shortest genome of the eight HBV genotypes (3182 nucleotides) [18].

11. Novel information of genotype D

In an analysis of the subgenotypes of D recently, it has been concluded that there are six, not eight subgenotypes. Subgenotypes D1–D6 can be distinguished by a separate cluster with high bootstrap support and amino acid signature. Subgenotype D3 and subgenotype D6 were reclassified as one sub-genotype D3, and genotype D/E instead of subgenotype was found to be genotype D/E. The D4 subgenottom may be an early substratum of early human intercontinental migration and may occur in indigenous peoples in Papua New Guinea and Australia and in a limited proportion of the Canadian Inuit people. In addition, a subgenotype D4 recombinant was same [18].

12. Genotype E

Genotype E is definitely the dominant genotype in West Africa and has very low intra-genotypic diversity suggesting that this genotype has spread only recently [21]. Genotype E strains specified of subtype ayw4, and all derived from West Africa apart from one strain which was derived from Madagascar. There were no subdivisions or specific amino acid substitutions distinguishing the strains from each other. All strains expressed Ser (140) also present in the genotype F. Study analysis of the complete genome of genotype E strains showed that the chimpanzee strain was not ancestral as compared with the human strains. This chimpanzee has probably also been inoculated with human serum at capture, since the majority of indigenous HBV strains from chimpanzees cluster separately from human strains [42].

Genotype E has the single Ayw 4 serologic subtype, which can be separated from A–D, F, H and I by the preS1 region’s deletion of 3 nucleotides. In West, Central Africa this genotype is endemic to a poor genetic diversity which has led to the recent appearance of more than 200 years. Contrary to the slavic trading subgenotype A1, genotype E is scarcely present outside Africa, with the exception of persons of African origin who further affirm its recently emerging after forced slavery migrations. A median developmental period of 130 years has been estimated using Bayesian inference, from the most recent common ancestor (tMRCA), this varies from a tMRCA predicted by others for 6,000 years. However, as previously indicated, genotype E may have occurred and recently been reintroduced in indigenous African populations. In persons with no experience of traveling to or from Africa, genotype E isolated from Pygmies and Khoi San, and in Colombia and India. The variation of the predicted genotype E age would be difficult to overcome without a precise determination of the nucleotide HBV substitution rate [42].

13. Genotype F

Genotype F has been isolated from Amerindian population in different countries [21]. Genotype F strains are subdivided into four subgenotypes. Subgenotype F1 particular F1a have been found in Alaska, El - Salvador, Guatemala, Costa Rica and Nicaragua; whereas F1b has been reported in Peru and Argentina. Strains of subgenotype F2 has been found in Costa Rica, Nicaragua, Venezuela and Brazil. Subgenoype F3 is found in Colombia and Venezuela and F4 in Bolivia and Argentina [21]. F1 and F2, each characterized by specific substitutions in the S gene product, Leu (45) and Thr (45), respectively. Subgenotype F1 was mainly formed by strains from Central America. F2 mainly containing strains from South America encompassed all strains from Venezuela and Colombia and the few strains from Polynesia and were characterized by an Asp (2) Glu substitution. Subtype adw4q– is alongside with adrq– the dominating subtypes in Polynesia. This supports a dual origin of its population, and the close relation of the Polynesian strain to strains from Colombia. Most of genotype F strains specified adw4 subtype. All had the Pro (178) Gln substitution assumed to abolish the expression of q. Some strains lacked the Pro (127) Leu substitution characteristic of genotype F and specified the putative subtype adw2q [42].

14. Genotype G

Genotype G is mostly detected in co-infection with other HBV genotypes with mostly genotype A [21]. Genotype G strains are originating from the USA, Mexico, and Europe [6] which are all specifying adw2 subtype. The genotype G strains shared two unique substitutions, Gln (51) Leu and Thr (63) Ile, were not found in any other genotype. The S gene products of the strains showed the highest similarity to those of genotype A. However, these strains showed a high divergence from the other HBV strains, when complete genomes were compared [42]. Genotype G strains have a 36 bp insertion immediately after the initiation codon of the C gene, increasing the size of HBcAg by (12 aa). This does not effect on Polymerase but a one codon deletion in the Pre-S1 region reduces both Pre-S1 and Pol by one aa [23].

Genotype G is characterized by the use, at the positions 2 and 28 in the precore/core region, of a 3′ nucleotide insert, 3′ of position 1905 and two translation stop codons which abrogate HBeAg. Only in the presence of other genotypes, most often genotype A, that may supply HBeAg in Trans, may chronic infection be identified. Sexual reproduction by males who have sex with men is a significant risk factor. Genotype G is not as diverse from genotype E with which the deletion of 3-nucleotide occurs in the central area and a special preS sequence. Since the African root of genotype G was not yet found in Africa [23].

15. Genotype H

All strains belonging to the genotype H derived from Nicaragua, Mexico, and California. These strains differ from genotype F strains by two unique substitutions, Val (l44) and Pro (45), as well as Ile (57),Thr (140), Phe (158), and Ala (224) [42, 45, 46].

16. Genotype I and its subgenotypes

In 2008, a study sequence of the whole genome (AB231908) of the Vietnamese male was suggested that it was closely linked to three previously identified Vietnamese ‘aberrant’ strains as well as to one of the 9th, I genotype. This was unacceptable since the average genetic divergence of these 4 genotype C strains was 7%, and the recombination study was not strong. The following sequences is derived from Laos, the tribe of the Idu Mishmi in North-East India, Canadian of Vietnamese origin, and China. At least 7.5 per cent of the nucleotide divergence between each of these sequences was with strong group bootstrap assistance, thus satisfying the genotype assignment criterion. Two subgenotypes I1 and I2, respectively, were identified with serological subtype’s adw 2 and ayw 2. This subgenotype isolation was challenged by the sequencing of additional Indian cluster strains in subgenotype I2 and by an estimated intersubgenotype differences of <4%. The intergroup divergences between subgenotype I1 and I2 were found to be 3,40 ± 0,30 percent (mean ± SD) below the 4 percent cut, if we analyzed all 19 genotype I genomes without indels in the GenBank. However, an exception should be made for subgenotype D1 and D2 due to the distinct serological subtypes. 4.1% between Laotian strain FJ023663 and the Indian strain EU835242 were the largest intergroup divergence. This genotype is endemic in a broad region of Asia for a long time because of the extensive geographical range. Genotype I is a recombinant of A/C/G genotypes and an indeterminate genotype which, when analyzed, is closely related to C genotype and the genotype A of polymerase genotype. The areas of genotype A and C are closely associated with A3s and C3s. In both Huh7 cells and acute hydrodynamic mouse infection, Genotype I has been functionally characterized. The two schemes also secreted genotype I at levels comparable to genotype A, genotype B and generic C and higher than generic D, but genotype A at levels related to genotype A and below B, C and D [42, 45, 46].

17. Genotype J

This strain had been isolated from one Japanese man who had long-term residence in Borneo with hepatocellular carcinoma (HCC). The entire non-human HBV genome clusters including gibbon isolates, orangutans, chimpanzees and gorillas. Their rates diverged 10.7–15.7% from other genotypes relative to 1,440 human and non-human HBV strains and did not demonstrate any indication that they were recombined. In the later study, Locarnini et al. concluded that genotype ‘J’ is actually a recombinant of genotype C and gibbon HBV in the S area, using additionally the gibbon/orangutan sequences for contrast. Therefore, while there is a strong intergroup divergency of genotype J, this will reflect propagation of cross species, detection and examination of additional sequences, until the presence of this last genotype can be verified. This requirement is defined for classification into separate genotype (Figure 5) [18].

Figure 5.

Global geographical distribution of HBV genotypes. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969325/pdf/40506_2016_Article_80.pdf.

Some studies have shown that different HBV genotypes and subgenotypes may cause differences in disease progression, response to antiviral treatment regimens and in clinical outcomes. Therefore, the accurate classification of HBV is important for clinical and etiological investigations [47].

18. HBV serotypes

The major classification of HBV subtype is sorted into 4 subtypes or serotypes (adr, adw, ayr, and ayw) [21, 45]. The molecular basis for this classification was variation at few sites in the S region. The ‘a’ determinant (aa 124–148) is the major antigenic determinant common and confers protection against all serotypes [41], while the d/y and w/r variations depend on Lys/Arg substitutions at residue (122) and (160) respectively [6]. If the amino acid at position (122) is Arg (122R) then the subtype is y, and if it is Lys (122 K) then the subtype is d. In the same way, (160R) defines the r subtype and (160 K) defines the w subtype. The four possible combinations define the major subtypes and additional amino acids contribute to immunogenicity. These subtypes can be further classified into (9) serotypes (adw2, adw4q-, adrq+, adrq-, ayw1, ayw2, ayw3, ayw4 and ayr). Epidemiologic studies found that the prevalence of these serotypes varies in different parts of the world. To date, there has been very little data on the clinical significance of HBV serotypes [45]. While the ability to detect HBsAg was of obvious importance for the safety of the blood supply, serotyping was useful for widely employed in clinical, virological, epidemiological studies, including studies of nosocomial and iatrogenic infections and intra-familial transmission, [23, 48].

Determinants w1/w2, w3 and w4 are classified by Pro, Thr or Leu substitutions at residue (127) respectively. w1 variation is distinguished by Arg122, Phe134 and/or Ala159 [49]. It has been found that the epitope in adw contains (18Val-19Pro), whereas these amino acids are replaced by hydrophilic residues Thr-Ser in the ayw1, 2, and 3 subtypes. As a consequence of these substitutions, the conformation of the epitopes, as predicted by 3D modeling and analysis of crystal structures, was drastically changed [50]. Cui and coworkers found that the serotype adw is based on Lys (120) and Lys (160). To a large extent, genotypes and subgenotypes have replaced the usage of serotypes. Most ayw serotypes are grouped in genotypes B and D [51]. The serotype ayw occurs in all genotypes except in ‘C’. Serotype adw is associated with all genotypes except D and E, whereas adr and ayr subtypes are encountered with genotype C [5]. There is no stringent correlation between phenotypic HBsAg markers and sequence variation outside the S gene but such a correlation between genetic and phenotypic markers is required for epidemiological studies [52].

19. Detect HBV genotype

Detection of HBV genotype is very important to clarify the pathogenesis, rout of infection and virulence of the virus. The HBV genotypes are variable that could potentially influence the outcome of chronic HBV and the success of antiviral therapy. HBV genotype testing has not yet been widely adopted in clinical laboratories. A variety of methods have been used, including whole or partial genome sequencing, PCR based restriction fragment length polymorphism (RFLP), genotype-specific PCR amplification, PCR plus hybridization, line probe assay, enzyme-linked immunoassay and serology. Whole-genome sequencing is the “gold standard,” and it is particularly accurate for detecting recombinant viruses [38, 53]. The common assays are:

19.1 INNO-LiPA

This reverse hybridization method has been developed by Innogenetics and is commercially available as INNO-LiPA. This method is easy to perform, very convenient, rapid method [54, 55], and suitable for detecting mixed genotype infections. First, HBV DNA is amplified by PCR using biotinylated primers complementary to a conserved sequence in the S/Pre-S ORF. The amplified biotinylated PCR products are then hybridized to probes immobilized onto membrane strips that detect genotype specific differences in the HBV polymerase gene domains B to C. After washing, alkaline phosphatase (ALP) - labeled streptavidin is added, followed by substrate (BCIP/NBT chromogen) that gives a purple/brown precipitate in the presence of ALP. The overall success rate is 98% [56]. These methods may fail to type all isolates and interpretation of results may be difficult particularly in the case of mixed genotype infections [55]. In addition, this assay is not suitable for large- scale surveys nor accurate to identify mixed infection [57].

19.2 HBV DNA-Chip assay

The whole HBV genome is amplified by a duplex PCR. The labeled PCR products were purified using a purification kit. Samples were hybridized on the HBV DNA-Chip prototype and stained with streptavidin-phycoerythrin conjugate on a GeneChip fluidics station 400. Finally, the HBV DNA-Chips were scanned on an HP Gene Array scanner and were analyzed by using DNA-Chip evaluation software. DNA-Chip technology is currently not used routinely in a clinical laboratory [58].

19.3 Nested-multiplex qPCR

A detection assay is used specific primers. This assay is greater accuracy in genotyping and greater sensitivity to identify mixed genotypes when compared to sequencing reactions. This method can be useful with large clinical scale and epidemiological studies, especially in regions with high prevalence of HBV infection [59].

19.4 Oligonucleotide microarray

Can determine genotypes A-G. The amplified products are heat-denatured and added to silylated slides, to which genotype-specific probes are immobilized [56].

19.5 Enzyme immunoassay (EIA)

This commercially assay is used with monoclonal antibodies raised against genotype specific epitopes in the Pre-S2. Although this assay may fail to type the HBV DNA in clinical samples due to the presence of mixtures of genotypes or low levels of HBsAg in the sample, it offers a convenient, serologically based assay, [55, 60]. EIAs were less sensitive than rapid assays [61]. ELISAs for HBsAg are generally considered more sensitive. It has been showed that the samples with low HBsAg/HBcAg ratios were much more likely to have undetectable Pre-S2 epitopes by the genotyping ELISA that used [31].

19.6 TaqMan-MGB probe

This assay has several advantages. On the one hand, conjugated MGB can improve the melting temperature of probe, thus increasing probe specificity. In addition, it permits shorter probes to be used (usually 13 to 18 nt). On the other hand, shorter probes make fluorescence and quencher closer A type-specific nested PCR assay established and applied for investigation of HBV genotype. The TaqMan technique is suitable for typing [40, 62, 63].

19.7 Line probe assay

This assay is detected sequence specific oligomers for each genotype are immortalized on a paper strip, to which PCR amplified test samples are hybridized (reverse hybridization) [5].

20. Limitations of using in-house assays

Many of limitations emerge when using in-house assays depending on the type of assay. It has often been suggested that in-house PCR assays suffer from problems with standardization, false positivity, or contamination, making them unsuitable for routine clinical diagnostic use [58]. The lack of an internal control does not allow to rule out false-negative results due to the presence of inhibitors to PCR amplification. The limit of detection and the upper limit of the dynamic range are approximates, as a lot more replicates and lot-to- lot testing would be necessary to verify these values.

One disadvantage of ELISA is that not all antibodies can be used monoclonal antibodies must be qualified as matched pairs, meaning they must recognize separate epitopes on the antigen so they do not hinder each other’s binding. Also, there is a limit to its sensitivity since the amplification is restricted by the amount of enzyme that can be conjugated to antibodies. Immunoreactivity of the antibody may be reduced by enzyme labeling, which in itself is an expensive and time-consuming process [64].

HBV genotyping based on complete genome sequences is an ideal methods, but sequencing is still expensive and not easy to carry out for large scale study. The developed precise PCR genotyping system using type-specific primers, allowing the identification of types A through F. This assay system may be useful for rapid and sensitive genotyping of the HBV genome either epidemiological, pathological, transmission studies and can be carried out in large scale. Mixed-genotype infection is very difficult to detect by direct sequencing. Since direct sequencing or Sanger sequencing can pick up mixed populations only at ratios above 20:80 simultaneous [24, 40, 47, 60, 65, 66].

20.1 PCR-RFLP

RFLP depends usually on PCR amplification of the S gene, restriction enzyme digestion, and separation of digested fragments by electrophoresis. A combination of different restriction enzymes has been used for RFLP, the choice of which has been determined according to the different HBV genotype sequences in GenBank. This method has been used to determine genotypes A–F. In 2004, Zeng et al. developed a modified RFLP technique based on the S gene allowing the detection of HBV genotypes A–H. In this method, two PCR rounds were undertaken prior to restriction enzyme digestion by five enzymes, namely StyI, BsrI, DpnI, HpaII, and EaeI. The method was compared with another RFLP method targeting the Pre-S1 region and the results were concordant in 96.8% [54].

RFLP typically relies on Sgene enhancement PCR, restrictive digestion of the enzyme, and electrophoresis isolation of the digested fragments. RFLP was used for a mixture of various restrictive enzymes, which were determined by the different HBV genotype sequences in GenBank. This approach is used in the A–F genotype determination. In 2004, Zeng et al. introduced a modified S-based RFLP technique that detects HBV A–H genotypes. In this process, five enzymes, StyI, BsrI, DpnI, Hpaii and EaeI, were used before the restriction of enzyme digestion. The method was contrasted with another RFLP method for the Pre-S1 field, with 96, 8 percent of the data.

Nested PCR-RFLP method for HBV genotyping is simple and inexpensive for clinical diagnostic in large scale. PCR-RFLP assay is more sensitive to identifying HBV viral populations [28]. This method can detect mixed genotypes and can determine subgenotypes in large population studies [56]. Toan et al. used the restriction enzyme Tsp509I to restrict patterns and predicted fragment sizes determined HBV genotypes, while Zeng et al. used five restriction enzymes, StyI, BsrI, DpnI, HpaII and EaeI were deemed to be suitable for yielding restriction patterns. These enzymes restrict at Per-S region (other study used EcoR1. This novel method would identify several relative advantages. Firstly, it can identify all eight HBV genotypes. Secondly, it is more accurate because it was based on analyzing many of the sequences deposited in GeneBank. Thirdly, a simple and inexpensive strategy can be adopted according to the most prevalent HBV genotypes in a particular geographical region. Moreover, this method can be useful in evaluating clinical, epidemiological and virological differences between genotypes [67]. Venegas used restriction enzymes Sau3A I, Bsr I or Hpa II to cut the DNA at S region. Vivekanandan and coworkers were used HinfI and Tsp 509I restriction enzymes but he found that genotypes could not be assigned for a small proportion of strains and this may be due to the presence of infection with multiple genotypes or with strains that have altered and or additional recognition sites for the restriction enzymes used in testing [68]. Neisi et al. used AvaII and mboI restriction enzymes but he resulted that the RFLP method cannot type mixed genotype infections. Other study used AvaII and DpnII [69, 70].

Badar used five restriction enzymes AlwI, EarI, HphI, NciI and NlaIV. He explained that the genotyping system can help to evaluate the etiological or clinical relevance of HBV genotypes and to predict the progression of liver disease or to investigate routes of infection [67]. Allen and coworkers described that the RFLP assay method has been commonly used for identifying known polymorphisms in DNA from many organisms or tissues and for detecting YMDD motif variations associated with in vitro lamivudine resistance patients. Moreover, although the RFLP assay was more sensitive in identifying HBV viral populations, one advantage of DNA sequencing over the RFLP that the DNA sequence provides information at sites other than at specific codons and continues to be useful in the detection of sequence variations at other sites for detecting quasi species. PCR-RFLP has some limitations. These include its retrospective nature and small sample size. Also, using a method is based on only a part of and not the entire of HBV genome [4].

Restriction fragment mass polymorphism (RFMP) is another method for detecting genotypes. Lee et al. utilized RFMP for HBV genotyping based on genotypic variations in the S gene, which is similar to RFLP. This method depends on restriction enzyme digestion of PCR products to produce genotype specific oligonucleotide fragments. The mass of the produced fragments is then determined using matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry. Other studies have reported the use of MALDI-TOF mass spectrometry for determination of YMDD (tyrosine–methionine–aspartate–aspartate–motif) mutations, which are linked to lamivudine (LAM) drug resistance [54].