Epidemiology, Genotypes and Subtypes

## **Chapter 1** Epidemiology of Hepatitis B Virus

*Cibangu Katamba and Onoya Onaluwa Philippe*

### **Abstract**

Hepatitis B virus (HBV) is a double-stranded DNA hepadnavirus. It is an important cause of acute 5and chronic hepatitis and hepatocellular carcinoma. Worldwide about 2 billion people show serological evidence of exposure and about 400 million have active infection. High prevalence areas include sub-Saharan Africa, China, and southeast Asia. HBV was known at onset as the etiology of what is called "serum hepatitis", this is the most common form of viral hepatitis transmitted parenterally. It is also a cause of both acute and chronic hepatitis of great significance. Hepatitis B virus has an incubation period that varies between 1 and 6 months. The clinical features of acute infection resemble those of the other viral hepatitides. Death from fulminant hepatitis occurs in about 1%. Following acute infection, there is either complete recovery (with long-term immunity) or persistent infection. The latter occurs in 5–10% infected adults, 30% infected children and 90% infants infected at birth; it is more common in the immunocompromised.

**Keywords:** hepatitis B, viral hepatitis, epidemiology, incidence, prevalence, distribution

#### **1. Introduction**

Hepatitis B Virus (HBV) is a double-stranded DNA hepadnavirus. It is an important cause of acute and chronic hepatitis and hepatocellular carcinoma. Worldwide about 2 billion people show serological evidence of exposure and about 400 million have active infection. High prevalence areas include sub-Saharan Africa, China, and Southeast Asia [1, 2].

HBV has remained a public health issue of global concern in the presence of multiple efforts to eradicate this viral disease through individualized and mass screening, education, and immunization programs [3]. The current estimates have shown that about 400 million individuals throughout the world have chronic HBV infection [1, 3]. Those infected with HBV have 15–40% risk of developing complications such as cirrhosis, hepatic failure, or hepatocellular carcinoma (HCC); and 15–25% of them have the risk of death from related HBV liver disease. About 60–80% of people are diagnosed with HCC and 500,000–1.2 million people die every year due to chronic HBV infection. It is established that HBV infection is the 10th leading cause of death globally.

HBV was known at onset as the etiology of what is called "serum hepatitis", this is the most common form of viral hepatitis transmitted parenterally. It is also a cause of both acute and chronic hepatitis of great significance [4]. Hepatitis B virus has an incubation period that varies between 1 and 6 months. The clinical features of acute infection resemble those of the other viral hepatitides. Death from fulminant hepatitis occurs in about 1%. Following acute infection, there is either

complete recovery (with long-term immunity) or persistent infection. The latter occurs in 5–10% infected adults, 30% infected children and 90% infants infected at birth; it is more common in the immunocompromised.

The prevalence of HBV infection varies widely [1–7], with rates ranging from 0.1–20% in different parts of the world. There is high prevalence of hepatitis B surface antigen (HBsAg) positivity rates above 8% in areas of high viral infection endemic prevalence such as the Far East, Sub-Sahara in Africa, the Amazon basin, and some parts of the Middle East. In these settings, there is presence of serologic evidence of prior hepatitis B virus infection anti-hepatitis B core antigen in most cases: positive anti-HBc or anti-HBs. Regions such as India, Japan, Middle East, Eastern & Southern Europe, parts of central Asia and south America are areas with intermediate chronic HBV infection prevalence (2–7% positive HBsAg). The regions with low chronic HBV infection prevalence (below 2percent positive HBsAg) include the USA, Australia, Southern South America, and Northern Europe. Throughout the world 45% of the global population live in high prevalence areas generally. People immigrating from high to low endemic regions have shown patterns that have a greater impact on the epidemiology of HBV; e.g., Migration of individuals from countries in South East Asia resulted in increased prevalence of chronic hepatitis B in the USA.

#### **2. Modes of transmission of hepatitis B virus**

We have two major modes of transmission of hepatitis B virus in the world [1–3]: perinatal and horizontal transmissions. The perinatal transmission occurs during child delivery from infected mothers to their babies, this mode of spread accounts for the majority of transmissions in the world. The second mode is horizontal transmission, that can occur through open wounds (cuts and scratches), blood transfusion, poor infection prevention practices to curve blood-borne infections in health facilities, sexual transmission and risky health behaviors such as piercing of the body, unsafe drugs injection, body tattoos and scarification using unsterilized instruments & other equipment. Developing chronic hepatitis B virus infection depends on the mode of spread of the virus, as the risk decreases with age at infection for susceptible individuals. About 90% of infections acquired during perinatal period will become chronical. Up to 20–60% of under 5 infections (1 to below 5 years of age) and 5–10% of adults and older children will develop chronic HBV infection.

Hepatitis B virus can be found in body fluids such as blood, saliva, vaginal fluids and menses, semen; and in less amount in breast milk, perspiration, urine, tears of infected people. HBV is easily spread through contact with body secretions, the virus resists to breakdown and can live long outside the human body. Heterosexual & other sexual activities and unsafe use of drugs by people who inject drugs account for most transmissions of the virus in regions with low prevalence.

Vertical or perinatal transmission of HBV is commonly observed in countries in the Far East Asian and Oceania regions. Mothers with high viral load have higher chances of transmitting the virus to their new born. Infection during child birth occurs in 5–20% of babies born to HBsAg positive, HBeAg-negative women. While most perinatal infections occur in babies born to chronically infected mothers, those with acute HBV infection in the 3rd trimester are also very likely to transmit the virus. Only less than 2% of perinatal transmissions occur in-utero.

The horizontal transmission during early childhood is significant in some regions in particular Sub-Saharan Africa, Alaska, and the Mediterranean, where perinatal transmission is less common as compared to Asia. There is lower prevalence of positive HBeAg correlates in mothers with less efficient spread during child

#### *Epidemiology of Hepatitis B Virus DOI: http://dx.doi.org/10.5772/intechopen.101097*

birth. Nonetheless, by the time the child is 10 years old, about 90% of children in rural Sub-Saharan regions of Africa will show evidence of past exposure to the virus. In the same population, HBeAg wean off in early in contrast to Asians, and most of them are HBeAg-negative by the reproductive age.

There is a possibility that these observations in epidemiologic variations are secondary to genotypic differences in hepatitis B virus. The accurate mode of spread in early childhood is not known, it is however thought that it occurs through blood and body secretions (not apparent) from family members or peers/playmates that inoculate the virus into cuts (scratches or abrasions) to the skin or other mucosal lesions.

The laboratory tests specific for HBV have revealed the fact that transmission through blood and blood products, and parenteral transmissions seem to be especially blood related. It is however important to note that infectivity does not solely appear by blood-to-blood contact. It has been observed that certain experimentations render the viral transmission by mouth is ineffective. The infection may be endemic in semi-closed and closed settings and mentally handicapped facilities. It is more common in urban settings among adults and those living in deprived socioeconomic states. Marked differences can be observed for the infection prevalence and the carrier states in various geographical areas and between people with different ethnic and socio-economic statuses.

Enough evidence exists to show the transmission of HBV by intimate and sexual contacts. People who are sexually promiscuous, especially those active homosexual males who change partners regularly, have very high chances of acquiring hepatitis B virus infection. The surface antigen for HBV has been detected in blood and various body secretions like semen, menses and vaginal fluids, saliva, breast milk (including colostrum) and serous fluids. These body fluids have been implicated in the transmission of the virus. Therefore, hepatitis through contact is of major importance. The virus may be accidentally transmitted from inoculation of small amount of blood or fluids contaminated with blood during medical or surgical interventions, vaccination with inadequately sterilized equipment such as needles and syringes, drug-injections, tattooing, piercing of ear and nose, acupuncture, razors, shared toothbrushes, towels and other linens contaminated with blood. Other factors related to transmission in specific climates in the tropics and warncountries are important to note; including ritual circumcision, blood-letting, repeated bites by bloodsucking arthropod vectors, traditional tattooing and scarification. However, findings on the role which biting insects play in the transmission of HBV are conflicting. HBsAg has been detected in multiple species of mosquito and bedbugs either trapped in the wild or fed experimentally on infected blood in laboratories. No convincing outcome for replication of hepatitis B virus in insect has been shown. Also, there is no evidence in epidemiology for mechanical transmission of HBV by insects.

HBsAg has been reported present in feces, urine and bile, often as a result of blood contamination. HBV is not known to be transmitted through fecal-oral mode and urine. Urine is not infectious unless it is blood contaminated. There is no evidence to show airborne transmission of the infection. Clustering of HBV can be seen in family settings but this is not associated with genetic factors and does not imply venereal or maternal transmission. The mechanisms of HBV intrafamilial transmission is yet to be established.

#### **3. Donation of tissues and blood**

As a standard of care, blood donors are now universally screened for hepatitis B with HBsAg test [1]. This had markedly reduced the risk of infection transmission

through transfusion. Countries around the world with negligible prevalence have also added anti-HBc tests to detect chronic carriers with low viral load who may not be detected with HBsAg test. These two tests decrease HBV infection rates to about 2.5–15.3 per million units of blood in settings with low prevalence rates. Developed countries like Canada, USA, Australia, Japan, etc. perform other more sensitive tests/nucleic acid tests additionally. However, the benefits in terms of incremental yield and other clinical advantages of nucleic acid tests over rapid tests (serologic) in these regions with low HBV prevalence, and the need for additional serologic tests, has not been established. Since about 90% of adults individuals have serologic evidence of ongoing or past HBV infection, anti-HBc is not to be used as a screening test in settings with high burden of hepatitis B. Therefore, in these settings, HBsAg is the only screening test being used. Nevertheless, in these settings, occult hepatitis B is seen in 3–30% of people with positive HBc and negative HBsAg. We now know that HBsAg-negative, positive hepatitis B DNA blood carries about 10% risk of transmission. In Taiwan, the risk of transmission of Hepatitis B infection through transfusion was approximated recently to be 100 per million units, with donor screening strategy using HBsAg test alone. This means that it is 7–40 times higher than in settings with low prevalence. The nucleic acid tests had a yield estimated to be at least 20 times higher in settings with high prevalence as compared to those with low prevalence, where it is currently in use. This has rendered it more cost saving per infection averted in these settings. Anti-HBc screening test is strategy is more cost-effective compared to nucleic acid tests in areas with lo prevalence despite the potential role of nucleic acid testing in these areas. The sensitivity and specificity of nucleic acid tests vary in most high prevalence settings. There is need to develop newer HBsAg assays with improved sensitivity to address this issue.

The other potential spread of occult or subclinical hepatitis B virus is from tissues and organs donation. Undetected viral load at time of tissue donation may be more frequent among tissue donors than blood donors according to estimates. To prevent infection following tissue or organ transplant, the easiest way is to exclude HBc-positive donors. This approach may not be practical in settings with high prevalence where most people have prior exposure to the virus. Consequently, the nucleic acid test is good additional measure to screening strategies for tissue donors to reduce the chances of transmission. The challenge, however, remains to reduce the turnaround time for results of nucleic acid test performed in clinical transplant facilities.

#### **4. Mother to child transmission**

HBV can be transmitted to infants born from carrier mothers during child labour and delivery. This is the single most important factor that determines the prevalence of HBV infection in some settings, especially the Southern parts of Eastern Asia and China. The chances of acquiring HBV infection in infant may approximate 90% and seem to be associated with ethnic groups. Pediatric infections are particularly important because a big number of these infants will be carriers. Hepatitis B infectivity is directly associated with the presence of high titres of HBsAg and/or HBeAg in the mother's blood stream. About95% of new born babies are infected around delivery time when HBeAg is present in their mother's circulation. The prevalence of HBeAg among mothers who carry the virus as well as mother to child' infectivity varies significantly in various settings and ethnic groups.

In South-East Asia, about 30–50% of HBsAg carrier mothers also carry HBeAg in their circulation. Perinatal transmissions are estimated to account for about 50% of the carriers in this population. These infections are frequent in babies born

#### *Epidemiology of Hepatitis B Virus DOI: http://dx.doi.org/10.5772/intechopen.101097*

from mother of West Asian and Afro-Caribbean decent. In the contrary, Caucasian women present fewer perinatal transmissions and carrier states. Mother to child transmission of infection and the carrier state patterns are different in regions such as Africa, where HBeAg is less common in carriers and the infection to their babies is frequently seen during the first 5 years of life resulting in horizontal transmission. The transmission of hepatitis B infection to infants born from non-carrier women by contact with other playmates who are infected from their carrier mothers is another mode of spread of the virus.

The considerable risk of hepatitis B infection during perinatal period from mothers with acute HBV infection is possible, especially during the 3rd trimester or within 2 months post-delivery. Transmission in-utero is not common, since HBV does not cross the intact placenta and the limited number of intrauterine infections are probably due to maternal blood leakage into the fetal blood stream associated with a tear in the placenta.

The exact mechanism of infection during perinatal period is not known but it is probable that this happens during delivery or shortly after birth due to maternal blood leakage into the fetal blood stream or the ingestion/inadvertent inoculation of maternal blood into the baby's circulation. The majority of infants infected during labour and delivery become chronic carriers.

HBeAg is a serologic marker for hepatitis B DNA viral load. Infections during perinatal period occur almost always in mothers who are positive for hepatitis B but can also occur in women who have very high viral load, hepatitis B virus DNA greater than 200,000 IU/ml in their blood. If the child is not immunized, his/her risk of acquiring hepatitis B virus during delivery is almost 100% when the mother has a positive HBeAg. The famous Taiwan study by Palmer Beasley as reported by Zuckerman [1] in the 1970s when there was no vaccine available showed that 85% of positive HBeAg mothers had their babies developing chronic infection vs only 32% of the negative ones. There is an approximated 90% risk of infants who acquired infection during perinatal period becoming chronically infected.

To reduce mother to child transmission of hepatitis B virus, incorporating the birth immunization dose into the HBV immunization schedule is the most effective strategy. This dose, if followed by two more doses, can significantly reduce the prevalence of chronic infection in babies born from positive HBeAg mothers by about 90%, and by about 100% from negative HBeAg mothers. The birth dose is particularly important in in settings were an important proportion of mothers with positive HBsAg and positive HBeAg at the same time. Such settings include the Pacific Islands, South East-Asia, and China. In these regions, if the birth dose is missed, HBV vaccine' effectiveness could reduce to about 50–75%. In other areas of the world such as Sub-Sahara in Africa, and Russia where less than 25% of pregnant mothers with a positive HBsAg have also positive HBeAg, the consequences of missing the birth dose are still significant but not as severe. The expended immunization program should include a dose of hepatitis B immunoglobulin at birth to babies born to mothers with positive HBsAg. This has the potential to reduce further the chances of transmission to below 5%. A randomized controlled trial by Beasley et al. demonstrated that the birth dose of hepatitis B immunoglobulin administered to babies born to mothers with both positive HBsAg and HBeAg lead to only 6% of these babies seroconverting to positive HBsAg as compared to 88% of babies in the placebo arm.

#### **5. Horizontal spread**

The horizontal spread of hepatitis B virus is very likely to lead to chronic state if it occurs in young children [1, 3, 6]. This was demonstrated in several studies

conducted before the HBV vaccine was made available. Research conducted in Senegal revealed that half the children who had horizontal transmission of HBV before they were 2 years old developed chronic infection. In another research on 1280 people who had hepatitis B virus negative sero-markers in rural Alaskan conducted in the 1970s revealed that 29% of children below 5 years old, out of 189 individuals who acquired the infection over a four-year period, had chronic hepatitis B against 16% of children between 5 and 10 years old, and only 8% for adults above 30 years old. The birth dose of hepatitis B vaccine together with the subsequent doses can reduce the acquisition of infection in the early months of life as well as prevent perinatal transmission in settings where the risk of chronicity through horizontal transmission of the infection is great.

Horizontal transmission, if it occurs in young children and some adults, this is because of high likelihood of infectious hepatitis B virus found on surfaces. The research conducted in Alaska many years ago, before the availability of viral DNA testing, the hepatitis B surface antigen was found in the environment on samples from table tops from school lunch room, toys, feeding bottles, and walls in houses where positive HBsAg individuals lived. Hepatitis B viral replication was possible at room temperature after at least 7 days. There is possibility that the virus can be spread through broken skin and mucosa from people with chronic infection on to surfaces, infecting thereby other people with open lesions. The horizontal transmission can also happen through non-sterile objects and procedures such as injections from healthcare providers or drug-injection, tattooing, scarification, sexual route, dialysis, emergency procedures, etc.

Young adults in the USA have increased horizontal transmission of HBV through unsafe drug-injection use in some places. There was 114% increase in the acute hepatitis B infection between 2006 and 2013 in West Virginia, Kentucky, and Tennessee. This increase was seen mostly in white populations between 30 and 39 years old who had a drug-injection history. In health care facilities, the outbreaks of hepatitis B can also increase the horizontal spread of the virus. The prevention of horizontal spread entails the combination of several measure such as education, good infection prevention and control practices, and immunization of household contacts to hepatitis B infected individuals and other people at high risk of HBV infection.

#### **5.1 Vaccination**

Hepatitis B vaccine is now available for over decades now. It is highly effective infection prevention measure among people at high risk of developing the disease [8]. The USA implemented universal immunization in 1991 and saw the incidence of acute infection decrease by 89% in adolescents and young children. With this exercise, the disparities in prevalence of chronic hepatitis B infection between races have reduced. Hepatitis B virus is endemic in Alaska, but following immunization, the incidence of new infections has markedly decreased. With this achievement, the incidence of hepatic cirrhosis and hepatocellular carcinoma is expected to reduce as well in the next few decades. Taiwan is one of the nations that adopted universal vaccination earlier. Its prevalence of positive surface antigen than was between 15–20% has reduced to 7% among adolescents and young children.

#### **6. Chronic carriers**

The concept "carrier state" is defined as persistence of HBsAg in blood circulation for more than 6 months, based on longitudinal researches. This state maybe

#### *Epidemiology of Hepatitis B Virus DOI: http://dx.doi.org/10.5772/intechopen.101097*

associated hepatic changes comprising minor damage in the nuclei of liver cells to persistent liver inflammation, chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. The integration of the HBV DNA may occur at several places or at unique site of the host genome in carriers of HBV with or without histological evidence of hepatic disease. The majority of carriers have HBsAg in their circulation with or without other markers of the viral infection (HBeAg, HBV DNA, DNA polymerase). The continued expression of HBsAg is suggestive of integrated viral DNA resultant. Some HBV carriers may have HBV DNA in their liver but with no surface antigen expression, this is called "latent viral infection".

There are a number of risk factors that have been established in accordance to the "carrier state". The carrier state is most common in male gender, it is more likely to follow infections acquired in childhood than those acquired in adulthood, it occurs most often in individuals with natural or acquired immune deficiencies. The carrier state develops in only about 5–10% of infections acquired in adult life.

Carriers' prevalence among adults who appear healthy, especially the blood donors vary by region. The global population can be grouped in to three regions by prevalence of hepatitis B virus infection:


With the advent of HIV/AIDS, the hepatitis B virus coinfection with HIV has become a major concern of late because of synergic negative effects of both viruses. HIV coinfection increase the chances of hepatic disease progression related to hepatitis B virus while hepatitis B coinfection augment antiretroviral therapy related liver toxicity.

#### **7. Distribution of hepatitis B by age**

It has been recognized two different patterns of hepatitis B infection by age distribution [1, 8, 9]. Individuals with high burden of HBV, infection is often acquired early during childhood. The highest infection and carrier rates are usually seen among children and young adults while the lowest is prevalent among older individuals. The HBeAg has been found more frequently in young carriers than in their adult counterparts. In contrast, HBe antibody is more common in older individuals. These results are in keeping with the possibility of young carriers being most infective.

#### *Hepatitis B*

In settings where this viral infection is not commonly observed, the highest prevalence of HBsAg occurs in populations between the age of 20 and 40 years old. The highest rates of hepatitis B infections are seen in populations at increased risk of contact with blood or blood products, e.g., health care workers, certain groups of patients, IV drug users, and male promiscuous homosexuals.

There is need to understand that the prevalence of hepatitis B infection, the age distribution of this infection, the carrier state. This change is drastic in some regions with the implementation of routine program of expanded hepatitis B vaccination.

#### **8. Hepatitis B genotypes**

There are six HBV genotypes grouped A-F based on phylogenetic analysis of complete viral genome classification [10, 11]. The most disseminated genotypes throughout the world are A and D. In contrast, B and C genotypes are restricted to East Asia, and E genotype to sub-Saharan Africa. The genotype F on the other hand is more diverse from other genotype classes and is seen in aboriginal Americans. All the genotype classes have a common immunodominant area on the surface antigen that is called "a determinant". This determinant span amino acids 124–147 and is hydrophilic. It is taught to be a form of two major and one minor loops with cysteine disulphide bonds. The "a" determinant target primarily the neutralizing antibodies induced by vaccination. The available hepatitis B vaccines have common major immunization response to "a" epitope with subsequent protection against all subtypes of HBV.

#### **9. Burden of HBV in developing countries**

HBV has an intermediate to high endemicity levels in developing countries [12–16]. Recently, the incidence of acute infection has decreased in several countries. The prevalence of chronic carriers of HBsAg has also decreased, this is as a result of the introduction of universal immunization coverage for hepatitis B virus in the 1990s. A few other countries are still not able to implement these interventions, especially in their rural and highly endemic regions. There is lack of sufficient information on the epidemiology of hepatitis B virus in many Eastern Europe and Latin-America countries.

#### **9.1 Epidemiology**

#### *9.1.1 Africa*

The entire Africa is known to be highly endemic continent for hepatitis B virus. The infection occurs with more than 8% hyperendemicity for chronic carriers of surface antigen in the general population in countries of Sub-Sahara like Nigeria, Cameroon, Burkina Faso, Gabon, and Namibia. Some other countries such as Zambia, Kenya, Senegal, Ivory Coast, Liberia, and Sierra Leone experience intermediate endemicity (2–8%). The following countries are considered to have low HBV endemicity (below 2%) in Africa: Morocco, Algeria, and Egypt.

Children in Africa are at high risk of acquiring hepatitis B infection. The hepatitis B markers seroconversion rates vary from 10.2–60.5% annually in Somalian children between 1 and 10 years old. The highest rates are seen in children with a low socio-economic situation. In South Africa, the highest rate of hepatitis B

#### *Epidemiology of Hepatitis B Virus DOI: http://dx.doi.org/10.5772/intechopen.101097*

infection in Children (5–6 years old) was 15.7%. The infection is often acquired by these children through parenteral horizontal transmission route from siblings and parents. Unsafe sharing of toiletries and sharpening, cutting, scraping or scratching instruments in the daily activities accounts for such a high horizontal transmission. In addition, cultural practices like scarification and tattooing and sexual promiscuity greatly increase the chance of hepatitis B infection. Hepatitis B transmission by transfusion of blood and blood products still occurs and is taught to have an epidemiological impact in some regions in Sub-Saharan countries.

#### *9.1.2 Asia*

The Arabian region or South-Western Asia accounts for 10% of territories in Asia. The Arabian Peninsula (Saudi Arabia, Bahrain, the Unites Arab Emirates, Oman, and Yemen included) together with Kuwait have a positive e antigen prevalence from 1.5 to above 8%. The HBsAg-positive prevalence in the Gaza Strip is 3.5% in the general population and 3.8% in blood donors.

Arab countries have implemented the WHO-recommended Expanded Program on Immunization, and hepatitis B virus immunization programs started in these countries have now covered a large proportion of their population. This has successfully reduced the hepatitis B virus endemicity.

Saudi Arabia is the first Arab country to adopt an HBV immunization program. It has seen a steady decline in positive surface antigen prevalence observed in children aged between 1 and 12 years, from 7% in 1989 to 0.31% in 1997 and zero% in 2008.

Cambodia is one of the western Pacific countries with the hepatitis B Virus prevalence at 4.6% in the adult population and 6% in blood donors. In this country, high anti-HBc rates have been reported (58.6% and 72.4%) in different studies, suggesting a principal role played in the past by horizontal transmission in childhood and adulthood.

China also started the universal HBV immunization program of newborn babies in 1992. In this country, the prevalence of surface antigen carriers decreased from 9.8% in 1992 to 7.18% in 2006. The immunization coverage rate at the end of 2005 was 20% lower in rural areas than in the urban areas, a difference that has steadily decreased in recent years. China has gone from a high to an intermediate endemicity level in a short period of time despite the suboptimal immunization coverage. The prevalence of anti-HBs was higher in fully immunized children (63.2–74.3%) than in non-immunized subjects (21.1–34.8%) because of the universal hepatitis B immunization campaign.

#### *9.1.3 Eastern Europe*

There are very few epidemiological studies conducted on hepatitis B virus infection in Eastern Europe which do not provide conclusive evidence on the spread of hepatitis B at the level of generalization of routine immunization in this large geographic region.

In a recent study from Bulgaria, positive surface antigen prevalence in persons below 20 years old, targeted by hepatitis B immunization, was significantly lower than that found in non-vaccinated persons aged over 20 (1% against 4.8%). The hepatitis B surface antigen-positive seroprevalence in the general population was 3.8% in studies performed in Bulgaria, 5.6% in Romania and from 4.4–13% in different studies in Serbia, with wide variations within single countries that reflect the different socio-economic conditions between rural and urban areas. In these studies, males showed higher rates of hepatitis B surface antigen positivity than females.

#### *9.1.4 Latin America*

The epidemiological information on hepatitis B virus is insufficient and in pieces in Latin America. About 7–12 million Latin Americans are carriers of hepatitis B chronic infection according to estimates. The rate of positive surface antigen individuals varies between countries, the highest being recorded in the 20–40 age groups possibly because of horizontal transmission. Recently, progress from intermediate to low endemicity levels have been registered in some tropical countries in Latin America such as Venezuela, Colombia, and Panama. Hepatitis B infection still provides a heavy socioeconomic burden in many developing nations despite universal immunization programs introduced in the 1990s. These programs need to be extended without fail to cover the rural areas in countries where hepatitis B vaccination is demonstrating its efficacy in reducing the transmission of the virus. Countries that are still unable to adopt a universal immunization program for newborn babies need to receive support from international health organizations to implement this.

#### **10. Burden of HBV in the United States**

There are approximately 700,000 to greater than 2 million people with chronic HBV infection in the USA. It is difficult to obtain accurate approximations of individuals burdened with chronic HBV infection in the world and in the USA in particular due the asymptomatic nature of the disease in most people infected with the virus [9]. This results in more people not diagnosed, passive surveillance, and underreporting. With the introduction of universal immunization in the USA, there is increased immunity among children and adolescents. Despite this progress, the number of adults infected with chronic hepatitis B has been increasing because of immigration of infected individuals from highly endemic settings. It is estimated that about 70% of hepatitis B infections in the USA are from foreign-born individuals. About 40,000–45,000 subjects from hepatitis B virus endemic settings (with chronic hepatitis B infection prevalence above 2%) immigrate to the USA legally. The total number of immigrants from Eastern Asia and Sub-Saharan Africa living in the USA is estimated above 3.9 million.

The National Health and Examination Survey (NHANES, 2011 & 2012) revealed that about 850,000 Americans are living with Chronic hepatitis B. Non-Hispanic individuals represent approximately 5% of the United States population. The oversampling of this group revealed that about half of all chronic hepatitis B infections (400,000) in the USA are seen among non-Hispanic Asians. The rates of acute hepatitis B infection have remained about 1 per 100,000 population since 2009 in the USA. These have been reported mainly from non-urban as compared to urban areas. The highest rate of acute hepatitis B infection in the USA is reported among African American adult populations. Of late, between 2006 and 2013, there was an increase in incidence of acute hepatitis B infection in Tennessee, West Virginia, and Kentucky, among white populations between the age of 30 and 39 years old who reported common risk factor such as drug-injection use.

*Epidemiology of Hepatitis B Virus DOI: http://dx.doi.org/10.5772/intechopen.101097*

#### **Author details**

Cibangu Katamba\* and Onoya Onaluwa Philippe Lusaka Provincial Health Office, Lusaka, Zambia

\*Address all correspondence to: patrickkatamba@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Epidemiology of Hepatitis B Virus DOI: http://dx.doi.org/10.5772/intechopen.101097*

DOI: 10.1128/CMR.00046-19. Available from: https://pubmed.ncbi.nlm.nih. gov/32102898/

[16] Sunbul M. Hepatitis B virus genotypes: Global distribution and clinical importance. World Journal of Gastroenterology. 2014;**20**(18):5427- 5434. DOI: 10.3748/wjg.v20.i18.5427. Available from: https://pubmed.ncbi. nlm.nih.gov/24833873/

#### **Chapter 2**

## Hepatitis B Virus, Genotypes and Subtypes

*Ali Adel Dawood*

#### **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.

**Keywords:** HBV, genotype, serotype, subtype, *Hepadnaviridae*

#### **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 [2–4].

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 [5–7]. 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,

#### **Figure 1.**

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

*Hepatitis B Virus, Genotypes and Subtypes DOI: http://dx.doi.org/10.5772/intechopen.100446*

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**).

#### **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

#### **Figure 2.**

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

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].

#### **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

#### **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=2ahUKEwiTsoLLsajwAhVSNOwKHXCvCuoQjJkEegQIAxA B&biw=1366&bih=658.*

**Figure 4.** *The architecture of a Dane particle. https://people.rit.edu/japfaa/infectious.html.*

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].

#### **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


#### **Table 1.**

*Differences between the main HBV genotypes [6].*

*Hepatitis B Virus, Genotypes and Subtypes DOI: http://dx.doi.org/10.5772/intechopen.100446*

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:

#### **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

*Hepatitis B Virus, Genotypes and Subtypes DOI: http://dx.doi.org/10.5772/intechopen.100446*

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%,

*Hepatitis B Virus, Genotypes and Subtypes DOI: http://dx.doi.org/10.5772/intechopen.100446*

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

#### **Figure 5.**

*Global geographical distribution of HBV genotypes. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969325/ pdf/40506\_2016\_Article\_80.pdf.*

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].

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 timeconsuming 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

#### *Hepatitis B Virus, Genotypes and Subtypes DOI: http://dx.doi.org/10.5772/intechopen.100446*

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].

### **Author details**

Ali Adel Dawood Department of Anatomy, College of Medicine, University of Mosul, Mosul, Iraq

\*Address all correspondence to: aad@uomosul.edu.iq

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Hepatitis B Virus, Genotypes and Subtypes DOI: http://dx.doi.org/10.5772/intechopen.100446*

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Section 2
