The Hepatitis B virus (HBV) is a major cause of liver disease, and according to the World Health Organization, around 257 million people live with Hepatitis B infection. The virus is a relatively ancient one in human history and has been infecting humans for at least 28,000 years. Previous studies have isolated HBV DNA from human skeletons dating from 800 to 7,000 years ago in Europe and Central Asia. Multiple types of this virus exist. Two types called HBV-A and HBV-D are present worldwide, with HBV-A being prevalent in Africa and in Europe, and HBV-D being very common in the Middle East and also in Europe.

Even though HBV has been infecting humans for millennia, there is little detailed knowledge of the how the disease spread among populations and geographical areas in the past. Due to few studies in this discipline, understanding of how the different types of HBV were dispersed and disseminated over time has remained patchy.

Now, Kostaki et al. analysed HBV-A and HBV-D DNA sequence data from present-day Hepatitis B patients to piece together a global map of historic spread of the virus. The results showed that HBV-D originated in North Africa and the Middle East, while HBV-A originated close to Africa and Europe and probably in the Middle East and Central Asia. HBV-A initially spread in Central Africa, after which it split into two separate pathways. The first spread to Sub-Saharan/eastern and southern Africa, with the other stretching to Sub-Saharan/eastern Africa. Much later, major regional transmissions happened from Africa to Brazil, Haiti and the Indian subcontinent, which are thought to be most likely due to the slave trade.

Uncovering the history of the spread of HBV and the human activities associated with it can help to inform public health strategies for avoiding similar situations happening again. These findings could be specifically useful in prevention of HBV in geographical areas where transmission is a high risk, ultimately helping to take steps toward eliminating HBV.

Hepatitis B virus (HBV) is the main cause of liver disease with an estimated number of 257 million people being chronically infected worldwide (Schweitzer et al., 2015). HBV belongs to the family Hepadnaviridae. The HBV genome consists of a partially double stranded DNA molecule approximately 3.2 kb long that replicates via an RNA intermediate. As a result of the activity of the error prone reverse transcriptase, HBV is characterized by a high degree of genetic heterogeneity and is classified into 9 genotypes (A-I) (Kramvis, 2014) and a putative 10th genotype J (Tatematsu et al., 2009), with an intergroup divergence of at least 7.5% across the complete genome. All genotypes, except for genotypes E and G, are further classified into subgenotypes with a mean genetic divergence of about 4% (Schaefer, 2007). These genotypes and subgenotypes can display complex ethnical and geographical distributions (Kramvis, 2014). Although genotypes D and A are omnipresent around the globe, genotype A prevails in Europe and Africa, with genotype D prevailing in Europe and the Middle East (Schaefer, 2007). Genotypes B and C are mostly found in Eastern Asia and Oceania, genotype E in Central and Western Africa, while genotypes F and H in Latin America and Alaska (Norder et al., 2004). Genotype D is considered to be pandemic (Kramvis, 2014). Phylogenetic analysis of genotype D showed separation into nine distinct clusters [subgenotypes D1, D2, D3, Recombino-subgenotype (RS)-D4, RS-D5, RS-D6, RS-D7, RS-D8 and RS-D9 (formerly ‘D4’, ‘D5’, ‘D6’, ‘D7’, ‘D8’ and ‘D9’)] with high bootstrap support (Pourkarim et al., 2010).

Subgenotype D1 is dominant in North Africa, Europe, Western Asia, Indonesia and Australia (Kramvis and Paraskevis, 2013; Bozdayi et al., 2005; Garmiri et al., 2011; Yousif and Kramvis, 2013), D2 in the United Kingdom, Albania, Northeastern Europe, Russia, Malaysia and Japan (Yousif and Kramvis, 2013; Tallo et al., 2008; Zehender et al., 2012) and D3 predominates in Serbia, South Africa and the United States of America (USA) (Yousif and Kramvis, 2013; De Maddalena et al., 2007; Lazarevic et al., 2007). RS-D4 (formerly ‘D4’) is found in Haiti, the Arctic and Oceania (Yousif and Kramvis, 2013; Norder et al., 2004), RS-D5 (formerly ‘D5’) circulates mainly in indigenous populations in India (Banerjee et al., 2006), RS-D6 (formerly ‘D6’) in Tunisia, Morocco and Madagascar (Kitab et al., 2011; Meldal et al., 2009). RS-D8 and RS-D9 (formerly ‘D8’ and ‘D9’) have been found in Niger (Abdou Chekaraou et al., 2010) and India (Ghosh et al., 2013), respectively. An updated classification of genotype D has been published (Pourkarim et al., 2010; Yousif and Kramvis, 2013; Pourkarim et al., 2014).

Genotype A is predominant in Northwestern Europe, North America and sub-Saharan Africa. Although seven HBV-A subgenotypes (A1–A7) have been described in the literature, a comprehensive analysis of this genotype has resulted in an updated classification (Pourkarim et al., 2010). Genotype A is thus classified into subgenotypes A1, A2, A4, and Quasi-subgenotype (QS)-A3, because the latter group of sequences does not meet the criteria for a subgenotype classification (Pourkarim et al., 2014). A1 can be found in sub-Saharan Africa (South Africa, Congo, Tanzania, Malawi, Kenya, Zimbabwe, Uganda, Somalia), and in areas outside Africa where there was historically forced migration as a result of the slave trade (Kramvis and Paraskevis, 2013) including South Asia (India, Philippines, Bangladesh, Nepal) and South America (Kramvis, 2014; Banerjee et al., 2006; Bowyer et al., 1997; Sugauchi et al., 2004). A2 is mostly found in Europe and North America (Norder et al., 2004; Bowyer et al., 1997). QS-A3 (formerly ‘A3’, ‘A4’, ‘A5’) is frequently found in Gabon (Makuwa et al., 2006) and Cameroon (‘A3’) (Kurbanov et al., 2005), in Mali (‘A4’), and in Nigeria (Olinger et al., 2006) and Haiti (‘A5’) (Andernach et al., 2009). A4 (formerly ‘A6’) has been found in African-Belgian patients (Pourkarim et al., 2010) and QS-A3 (formerly ‘A7’) is circulating in Rwanda and Cameroon (Hübschen et al., 2011).

The clinical manifestation of HBV infection differs between individuals infected with genotype A compared to those infected with genotype D because of different molecular characteristics of these genotypes, especially in the precore region of the HBV genome. HBeAg-negative G1896A mutant chronic hepatitis B predominates in areas where genotype D prevails (Hadziyannis, 2011) because 1858T is positively associated with genotype D and negatively associated with genotype A, which has 1858C (Kramvis et al., 2008) and thus genotype A cannot develop precore G1896A. However, subgenotype A1 but not A2, has alternative mechanisms, which result in HBeAg-negativity (Kramvis, 2016). Consequently, subgenotype A2 shows the highest frequency of HBe-Ag-positivity compared to A1 and genotype D and this is highly statistically significant in individuals younger than 29 years (Tanaka et al., 2004). This means that subgenotype A2 has a longer high replicative, low inflammatory phase compared to subgenotype A1 and genotype D. A limited number of studies have shown that subgenotype A1, and perhaps genotype D are associated with an increased risk of developing serious complications of HBV in comparison with subgenotype A2 (McMahon, 2009; Kew et al., 2005; Gopalakrishnan et al., 2013), which is characterized by chronic HBV infection and sexual transmission (Hadziyannis, 2011; Araujo et al., 2011). Multivariate logistic regression analysis revealed only genotype A was independently associated with viral persistence following acute hepatitis B (Japanese AHB Study Group et al., 2014). Patients infected with genotype A respond better to interferon-based therapy compared to patients infected with genotype D (Lin and Kao, 2010; Kramvis and Kew, 2005). Although overall no significant difference in response of the different genotypes/subgenotypes to nucleos(t)ide analogue therapy has been found (Lin and Kao, 2010), response to adefovir may be lower in patients infected with subgenotype A2 because of the presence of L217R polymorphism in the S region (Bottecchia et al., 2008).

The epidemiological history of the HBV-D and HBV-A genotypes remains unclear because of the scarcity of relevant studies. In this study, we estimated the levels of regional clustering for HBV-D and HBV-A, in order to shed light on how these genotypes have been disseminated among geographic regions and countries over time. We also estimated their putative geographic origin and major dispersal pathways over the course of genotype A and D infection.

We used the maximum likelihood method with bootstrap evaluation to reconstruct the phylogeny of globally sampled, full-length, non-recombinant sequences of HBV genotypes D and A. The description of the spatial characteristics of viral dispersal plays a pivotal role in understanding the history of the respective infections and to make hypotheses about the parameters potentially associated with the observed dispersal patterns. We also performed phylogeographic analysis to estimate the origin of HBV lineages and the pathways of dispersal over the course of the genotype A and D infections.

Previous studies supported the ‘Out of Africa’ model for the origin and expansion of HBV by providing evidence of HBV/Humans co-expansion and co-development of their population sizes 22,000–47,100 years ago (Paraskevis et al., 2013), while estimating the origin of HBV in humans at 34,100 (27,600–41,300) years ago (Paraskevis et al., 2015). The ancient origin of HBV has been also confirmed by analysis of HBV sequences from two 16th century mummies and two recent studies, which detected HBV from ancient DNA samples, ranging in age from approximately 800 to 4,500 years (Mühlemann et al., 2018) and 1,000 to 7,000 years ago (Krause-Kyora et al., 2018), showed that the HBV infections were present for at least 7,000 years (Patterson Ross et al., 2018; Kahila Bar-Gal et al., 2012). As the major genotypes have probably originated before and during the onset of Neolithic and the subgenotypes during the later Neolithic period, the majority of HBV diversity has been accumulated as a result of dispersals following the antecedent ‘Out of Africa’ population migrations, which hosted and conveyed the parental HBV strains (Kramvis, 2014; Locarnini et al., 2013; Zehender et al., 2014).

It is important to note that rapid human population expansion occurred outside of Africa, following a radial pattern across the Eurasian continents. More specifically, Central Asia served as a node for the dispersal of Humans towards North Africa, the Eastern Mediterranean and South Europe and Southeast Asia before reaching the Americas (Henn et al., 2012). HBV follows the same routes, with serial founder events creating the major genotypes, which in turn become prevalent in a compartmental pattern of dispersal around the globe (Paraskevis et al., 2013, 2015). However, it is expected that regional monophyletic clustering abundance would not be identical and would be dependent on the levels of host mobility. For example, regional monophyletic clustering is found in geographic areas, which remained isolated for a long time (e.g. subgenotype B6 in the Arctic, subgenotypes C3, C4, RS-D4 in Oceania and the Pacific) (Kowalec et al., 2013; Osiowy et al., 2011; Lusida et al., 2008; Thedja et al., 2011) suggesting that the HBV dispersal was the result of onward transmissions of a single or few strains introduced in the population(s) in the past (Paraskevis et al., 2013). High proportions of strains not belonging to monophyletic clusters suggest significant population mobility giving rise to infections; whereas dominant regional monophyletic clustering suggests that an epidemic has been introduced from a single or few restricted sources (as a result of limited human and viral mobility).

In the present study, considerable differences regarding the patterns of dispersal and the regions of clustering of HBV genotypes D and A around the globe were identified. For HBV-D, we found low levels of regional clustering for North Africa/Middle East, suggesting high levels of viral mobility. This is in line with our previous observations, about the co-expansion of HBV with its host and the central role of North Africa and the Middle East regions as hubs for human expansion and consequent dissemination and genetic shuffling of genotype D (Paraskevis et al., 2013). Notably we also found that this area provides the putative origin for genotype D. This conclusion is supported by the finding that the location of the HBV sequence that clustered at the root of genotype D and sampled 2,300 years ago, was in Central Asia (data not shown) (Mühlemann et al., 2018). On the other hand, regarding Greenland and New Zealand, we found almost 100% monophyletic clustering suggesting that the genotype D infection in these areas were because of onward transmissions among the local population(s) and not due to introduction from recent human migrations into these areas. The high levels of monophyly clustering in East Asia (Japan, China) and North Africa (Tunisia) suggest a limited number of genotype D introductions in these areas. The high viral genetic diversity for these areas supports the hypothesis that the founder strains were not introduced because of recent human migrations following globalization during the 20th century. In contrast, India showed a low level of regional clustering suggesting a highly mobile infection. Moreover, the phylogeographic trees suggest that virus has been disseminated across different populations, probably as a result of extensive human mobility, at different time periods.

Our analysis revealed that HBV-A shows a strong pattern of regional dispersal with both macro- or micro-levels of clustering. Macro-level clustering showed that genotype A can be further grouped into two major clusters (African and European). These clusters correspond to subgenotype A1 in Africa and subgenotype A2 outside of Africa (Kramvis and Paraskevis, 2013). The putative origin of genotype A was probably in the Middle East/Central Asia and thereafter followed two distinct routes of dispersal, one within Africa and a second to Western Europe. The hypothesis that the putative origin of genotype A was in the Middle East/Central Asia was further supported by the recent analysis of HBV ancestral genotype A sequences in ancient samples (Mühlemann et al., 2018). Specifically, the location of two HBV sequences, which clustered at the root of the genotype A and sampled 4,000 years ago, was in Central Asia (data not shown) (Mühlemann et al., 2018). Supporting the results of our previous study, where we analysed subgenomic preS/S region, HBV-A strains from sub-Saharan Africa were the source for Caribbean, Latin America and South Asia following recent human mobility, as a result of the historical forced migration of the slave trade in the 16th – 19th centuries (Kramvis and Paraskevis, 2013). Similarly, from group II different regional clusters were detected for Western Europe and Latin America. Notably, the HBV-A in the USA had a Western European origin. This contrasts with Caribbean and some areas in Latin America, where HBV originated in Africa. Regional dispersal was dominant in Panama, Cameroon, South Africa and Rwanda.

The African dispersal pathways had, probably, a sub-Saharan origin from Central Africa and thereafter moving southwards. Introduction of HBV in Europe was the result of a founder effect occurring later than in Africa. The origin of parental European strains is missing. Notably genotype A has been introduced to Haiti, Brazil and the Indian subcontinent as a result of slave trade (Kramvis and Paraskevis, 2013).

In the current study, we analyzed the dispersal patterns of two of the most globally disseminated HBV genotypes. To our knowledge, this is one of the few studies showing the dispersal pathways based on the phylogeographic analysis of all full-length available data for genotypes A and D. The highest levels of clustering for genotype A suggest limited viral mobility at the earliest phase of this infection. Thereafter regional transmissions remained dominant as supported by high levels of monophyly. In contrast to HBV-A, for the exception of Tunisia, limited regional dispersal was found for genotype D in North Africa/Middle East, while this region acted as its putative source. Limited monophyly for these areas was observed at micro-level clustering as well suggesting high levels of mobility over the course of the infection. These findings can be explained by the fact that the area acted as a source for HBV-D but also the areas of the earliest dispersal showed high levels of human mobility for a number of reasons including the expansion of agriculture during the Neolithic revolution, the development of modern civilizations and the existence of major trade routes (Diamond and Bellwood, 2003). In contrast, during the same period, human mobility was relatively limited in sub-Saharan Africa, thus leading to regional dispersal of HBV-A. Our findings are corroborated by the recently published analysis of HBV from ancient samples (Mühlemann et al., 2018; Krause-Kyora et al., 2018), which show that HBV was already circulating in humans at least 7,000 years ago and, in agreement with our analyses, place the putative origin of genotypes A and D in the Middle East/Central Asia.

Our study has several limitations mostly related to potential sampling bias of HBV sequences available in the databases and the lack of information about country of birth or the immigration status of patients for whom HBV sequences were included in the current study. However, we do not expect that these limitations affect the levels of monophyly per country and the proposed dispersal pathways of the genotypes A and D.

In conclusion, the observed differences of the dispersal patterns and the levels of regional clustering between HBV-D and HBV-A around the globe, probably portray the impact of the prehistoric human activities on the evolution of this pathogen, but also highlight the importance of co-evolution of the host in phylogenetic reconstructions of slowly evolving pathogens such as HBV (Paraskevis et al., 2015).

All data (sequence alignments and additional pieces of information related to the accession numbers of sequences and their sampling areas) are available at Dryad (doi: 10.5061/dryad.bt4q242).

1. 1. Kostaki E
   2. Karamitros T
   3. Stefanou G
   4. Mamais I
   5. Angelis K
   6. Hatzakis A
   7. Kramvis A
   8. Paraskevis D

   (2018) Data from: Unravelling the history of hepatitis B virus genotypes A and D infection using a full-genome phylogenetic and phylogeographic approach

   Available at Dryad Digital Repository under a CC0 Public Domain Dedication.

   http://dx.doi.org/10.5061/dryad.bt4q242

1. 1. Abdou Chekaraou M
   2. Brichler S
   3. Mansour W
   4. Le Gal F
   5. Garba A
   6. Dény P
   7. Gordien E

   (2010) A novel hepatitis B virus (HBV) subgenotype D (D8) strain, resulting from recombination between genotypes D and E, is circulating in Niger along with HBV/E strains

   Journal of General Virology 91:1609–1620.

   https://doi.org/10.1099/vir.0.018127-0

   • PubMed
   • Google Scholar
2. 1. Alcantara LC
   2. Cassol S
   3. Libin P
   4. Deforche K
   5. Pybus OG
   6. Van Ranst M
   7. Galvão-Castro B
   8. Vandamme AM
   9. de Oliveira T

   (2009) A standardized framework for accurate, high-throughput genotyping of recombinant and non-recombinant viral sequences

   Nucleic Acids Research 37:W634–W642.

   https://doi.org/10.1093/nar/gkp455

   • PubMed
   • Google Scholar
3. 1. Andernach IE
   2. Nolte C
   3. Pape JW
   4. Muller CP

   (2009) Slave trade and hepatitis B virus genotypes and subgenotypes in Haiti and Africa

   Emerging Infectious Diseases 15:1222–1228.

   https://doi.org/10.3201/eid1508.081642

   • PubMed
   • Google Scholar
4. 1. Arankalle VA
   2. Gandhi S
   3. Lole KS
   4. Chadha MS
   5. Gupte GM
   6. Lokhande MU

   (2011) An outbreak of hepatitis B with high mortality in India: association with precore, basal core promoter mutants and improperly sterilized syringes

   Journal of Viral Hepatitis 18:e20–e28.

   https://doi.org/10.1111/j.1365-2893.2010.01391.x

   • PubMed
   • Google Scholar
5. 1. Araujo NM
   2. Waizbort R
   3. Kay A

   (2011) Hepatitis B virus infection from an evolutionary point of view: how viral, host, and environmental factors shape genotypes and subgenotypes

   Infection, Genetics and Evolution 11:1199–1207.

   https://doi.org/10.1016/j.meegid.2011.04.017

   • Google Scholar
6. 1. Banerjee A
   2. Kurbanov F
   3. Datta S
   4. Chandra PK
   5. Tanaka Y
   6. Mizokami M
   7. Chakravarty R

   (2006) Phylogenetic relatedness and genetic diversity of hepatitis B virus isolates in Eastern India

   Journal of Medical Virology 78:1164–1174.

   https://doi.org/10.1002/jmv.20677

   • PubMed
   • Google Scholar
7. 1. Bottecchia M
   2. Madejón A
   3. Sheldon J
   4. García-Samaniego J
   5. Barreiro P
   6. Soriano V

   (2008) Hepatitis B virus genotype A2 harbours an L217R polymorphism which may account for a lower response to adefovir

   Journal of Antimicrobial Chemotherapy 62:626–627.

   https://doi.org/10.1093/jac/dkn207

   • PubMed
   • Google Scholar
8. 1. Bowyer SM
   2. van Staden L
   3. Kew MC
   4. Sim JG

   (1997) A unique segment of the hepatitis B virus group A genotype identified in isolates from South Africa

   Journal of General Virology 78:1719–1729.

   https://doi.org/10.1099/0022-1317-78-7-1719

   • PubMed
   • Google Scholar
9. 1. Bozdayi G
   2. Türkyilmaz AR
   3. Idilman R
   4. Karatayli E
   5. Rota S
   6. Yurdaydin C
   7. Bozdayi AM

   (2005) Complete genome sequence and phylogenetic analysis of hepatitis B virus isolated from Turkish patients with chronic HBV infection

   Journal of Medical Virology 76:476–481.

   https://doi.org/10.1002/jmv.20386

   • PubMed
   • Google Scholar
10. 1. De Maddalena C
    2. Giambelli C
    3. Tanzi E
    4. Colzani D
    5. Schiavini M
    6. Milazzo L
    7. Bernini F
    8. Ebranati E
    9. Cargnel A
    10. Bruno R
    11. Galli M
    12. Zehender G

    (2007) High level of genetic heterogeneity in S and P genes of genotype D hepatitis B virus

    Virology 365:113–124.

    https://doi.org/10.1016/j.virol.2007.03.015

    • PubMed
    • Google Scholar
11. 1. Diamond J
    2. Bellwood P

    (2003) Farmers and their languages: the first expansions

    Science 300:597–603.

    https://doi.org/10.1126/science.1078208

    • PubMed
    • Google Scholar
12. 1. Garfein RS
    2. Bower WA
    3. Loney CM
    4. Hutin YJ
    5. Xia GL
    6. Jawanda J
    7. Groom AV
    8. Nainan OV
    9. Murphy JS
    10. Bell BP

    (2004) Factors associated with fulminant liver failure during an outbreak among injection drug users with acute hepatitis B

    Hepatology 40:865–873.

    https://doi.org/10.1002/hep.20383

    • PubMed
    • Google Scholar
13. 1. Garmiri P
    2. Rezvan H
    3. Abolghasemi H
    4. Allain JP

    (2011) Full genome characterization of hepatitis B virus strains from blood donors in Iran

    Journal of Medical Virology 83:948–952.

    https://doi.org/10.1002/jmv.21772

    • PubMed
    • Google Scholar
14. 1. Ghosh S
    2. Banerjee P
    3. Deny P
    4. Mondal RK
    5. Nandi M
    6. Roychoudhury A
    7. Das K
    8. Banerjee S
    9. Santra A
    10. Zoulim F
    11. Chowdhury A
    12. Datta S

    (2013) New HBV subgenotype D9, a novel D/C recombinant, identified in patients with chronic HBeAg-negative infection in Eastern India

    Journal of Viral Hepatitis 20:209–218.

    https://doi.org/10.1111/j.1365-2893.2012.01655.x

    • PubMed
    • Google Scholar
15. 1. Ghosh S
    2. Mondal RK
    3. Banerjee P
    4. Nandi M
    5. Sarkar S
    6. Das K
    7. Santra A
    8. Banerjee S
    9. Chowdhury A
    10. Datta S

    (2012) Tracking the naturally occurring mutations across the full-length genome of hepatitis B virus of genotype D in different phases of chronic e-antigen-negative infection

    Clinical Microbiology and Infection 18:E412–E418.

    https://doi.org/10.1111/j.1469-0691.2012.03975.x

    • PubMed
    • Google Scholar
16. 1. Gopalakrishnan D
    2. Keyter M
    3. Shenoy KT
    4. Leena KB
    5. Thayumanavan L
    6. Thomas V
    7. Vinayakumar K
    8. Panackel C
    9. Korah AT
    10. Nair R
    11. Kramvis A

    (2013) Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala, India

    World Journal of Gastroenterology 19:9294–9306.

    https://doi.org/10.3748/wjg.v19.i48.9294

    • PubMed
    • Google Scholar
17. 1. Hadziyannis SJ

    (2011) Natural history of chronic hepatitis B in Euro-Mediterranean and African countries

    Journal of Hepatology 55:183–191.

    https://doi.org/10.1016/j.jhep.2010.12.030

    • PubMed
    • Google Scholar
18. 1. Hass M
    2. Hannoun C
    3. Kalinina T
    4. Sommer G
    5. Manegold C
    6. Günther S

    (2005) Functional analysis of hepatitis B virus reactivating in hepatitis B surface antigen-negative individuals

    Hepatology 42:93–103.

    https://doi.org/10.1002/hep.20748

    • PubMed
    • Google Scholar
19. 1. Henn BM
    2. Cavalli-Sforza LL
    3. Feldman MW

    (2012) The great human expansion

    PNAS 109:17758–17764.

    https://doi.org/10.1073/pnas.1212380109

    • PubMed
    • Google Scholar
20. 1. Hübschen JM
    2. Mbah PO
    3. Forbi JC
    4. Otegbayo JA
    5. Olinger CM
    6. Charpentier E
    7. Muller CP

    (2011) Detection of a new subgenotype of hepatitis B virus genotype A in Cameroon but not in neighbouring Nigeria

    Clinical Microbiology and Infection 17:88–94.

    https://doi.org/10.1111/j.1469-0691.2010.03205.x

    • PubMed
    • Google Scholar
21. 1. Japanese AHB Study Group
    2. Ito K
    3. Yotsuyanagi H
    4. Yatsuhashi H
    5. Karino Y
    6. Takikawa Y
    7. Saito T
    8. Arase Y
    9. Imazeki F
    10. Kurosaki M
    11. Umemura T
    12. Ichida T
    13. Toyoda H
    14. Yoneda M
    15. Mita E
    16. Yamamoto K
    17. Michitaka K
    18. Maeshiro T
    19. Tanuma J
    20. Tanaka Y
    21. Sugiyama M
    22. Murata K
    23. Masaki N
    24. Mizokami M

    (2014) Risk factors for long-term persistence of serum hepatitis B surface antigen following acute hepatitis B virus infection in japanese adults

    Hepatology 59:89–97.

    https://doi.org/10.1002/hep.26635

    • PubMed
    • Google Scholar
22. 1. Kahila Bar-Gal G
    2. Kim MJ
    3. Klein A
    4. Shin DH
    5. Oh CS
    6. Kim JW
    7. Kim TH
    8. Kim SB
    9. Grant PR
    10. Pappo O
    11. Spigelman M
    12. Shouval D

    (2012) Tracing hepatitis B virus to the 16th century in a Korean mummy

    Hepatology 56:1671–1680.

    https://doi.org/10.1002/hep.25852

    • PubMed
    • Google Scholar
23. 1. Kew MC
    2. Kramvis A
    3. Yu MC
    4. Arakawa K
    5. Hodkinson J

    (2005) Increased hepatocarcinogenic potential of hepatitis B virus genotype A in Bantu-speaking sub-saharan Africans

    Journal of Medical Virology 75:513–521.

    https://doi.org/10.1002/jmv.20311

    • PubMed
    • Google Scholar
24. 1. Kitab B
    2. El Feydi AE
    3. Afifi R
    4. Derdabi O
    5. Cherradi Y
    6. Benazzouz M
    7. Rebbani K
    8. Brahim I
    9. Salih Alj H
    10. Zoulim F
    11. Trepo C
    12. Chemin I
    13. Ezzikouri S
    14. Benjelloun S

    (2011) Hepatitis B genotypes/subgenotypes and MHR variants among moroccan chronic carriers

    Journal of Infection 63:66–75.

    https://doi.org/10.1016/j.jinf.2011.05.007

    • PubMed
    • Google Scholar
25. 1. Kowalec K
    2. Minuk GY
    3. Børresen ML
    4. Koch A
    5. McMahon BJ
    6. Simons B
    7. Osiowy C

    (2013) Genetic diversity of hepatitis B virus genotypes B6, D and F among circumpolar indigenous individuals

    Journal of Viral Hepatitis 20:122–130.

    https://doi.org/10.1111/j.1365-2893.2012.01632.x

    • PubMed
    • Google Scholar
26. 1. Kramvis A
    2. Arakawa K
    3. Yu MC
    4. Nogueira R
    5. Stram DO
    6. Kew MC

    (2008) Relationship of serological subtype, basic core promoter and precore mutations to genotypes/subgenotypes of hepatitis B virus

    Journal of Medical Virology 80:27–46.

    https://doi.org/10.1002/jmv.21049

    • PubMed
    • Google Scholar
27. 1. Kramvis A
    2. Kew MC

    (2005) Relationship of genotypes of hepatitis B virus to mutations, disease progression and response to antiviral therapy

    Journal of Viral Hepatitis 12:456–464.

    https://doi.org/10.1111/j.1365-2893.2005.00624.x

    • PubMed
    • Google Scholar
28. 1. Kramvis A
    2. Paraskevis D

    (2013) Subgenotype A1 of HBV--tracing human migrations in and out of Africa

    Antiviral Therapy 18:513–521.

    https://doi.org/10.3851/IMP2657

    • PubMed
    • Google Scholar
29. 1. Kramvis A

    (2014) Genotypes and genetic variability of hepatitis B virus

    Intervirology 57:141–150.

    https://doi.org/10.1159/000360947

    • PubMed
    • Google Scholar
30. 1. Kramvis A

    (2016) The clinical implications of hepatitis B virus genotypes and HBeAg in pediatrics

    Reviews in Medical Virology 26:285–303.

    https://doi.org/10.1002/rmv.1885

    • PubMed
    • Google Scholar
31. 1. Krause-Kyora B
    2. Susat J
    3. Key FM
    4. Kühnert D
    5. Bosse E
    6. Immel A
    7. Rinne C
    8. Kornell SC
    9. Yepes D
    10. Franzenburg S
    11. Heyne HO
    12. Meier T
    13. Lösch S
    14. Meller H
    15. Friederich S
    16. Nicklisch N
    17. Alt KW
    18. Schreiber S
    19. Tholey A
    20. Herbig A
    21. Nebel A
    22. Krause J

    (2018) Neolithic and medieval virus genomes reveal complex evolution of hepatitis B

    eLife 7:e36666.

    https://doi.org/10.7554/eLife.36666

    • PubMed
    • Google Scholar
32. 1. Kumar S
    2. Stecher G
    3. Tamura K

    (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets

    Molecular Biology and Evolution 33:1870–1874.

    https://doi.org/10.1093/molbev/msw054

    • PubMed
    • Google Scholar
33. 1. Kurbanov F
    2. Tanaka Y
    3. Fujiwara K
    4. Sugauchi F
    5. Mbanya D
    6. Zekeng L
    7. Ndembi N
    8. Ngansop C
    9. Kaptue L
    10. Miura T
    11. Ido E
    12. Hayami M
    13. Ichimura H
    14. Mizokami M

    (2005) A new subtype (subgenotype) Ac (A3) of hepatitis B virus and recombination between genotypes A and E in Cameroon

    Journal of General Virology 86:2047–2056.

    https://doi.org/10.1099/vir.0.80922-0

    • PubMed
    • Google Scholar
34. 1. Lazarevic I
    2. Cupic M
    3. Delic D
    4. Svirtlih NS
    5. Simonovic J
    6. Jovanovic T

    (2007) Distribution of HBV genotypes, subgenotypes and HBsAg subtypes among chronically infected patients in Serbia

    Archives of Virology 152:2017–2025.

    https://doi.org/10.1007/s00705-007-1031-0

    • PubMed
    • Google Scholar
35. 1. Lin CL
    2. Kao JH

    (2010) Clinical implications of hepatitis B virus variants

    Journal of the Formosan Medical Association 109:321–325.

    https://doi.org/10.1016/S0929-6646(10)60059-9

    • PubMed
    • Google Scholar
36. 1. Locarnini S
    2. Littlejohn M
    3. Aziz MN
    4. Yuen L

    (2013) Possible origins and evolution of the hepatitis B virus (HBV)

    Seminars in Cancer Biology 23:561–575.

    https://doi.org/10.1016/j.semcancer.2013.08.006

    • PubMed
    • Google Scholar
37. 1. Lole KS
    2. Bollinger RC
    3. Paranjape RS
    4. Gadkari D
    5. Kulkarni SS
    6. Novak NG
    7. Ingersoll R
    8. Sheppard HW
    9. Ray SC

    (1999)

    Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination

    Journal of Virology 73:152–160.

    • PubMed
    • Google Scholar
38. 1. Lusida MI
    2. Nugrahaputra VE
    3. Soetjipto
    4. Handajani R
    5. Nagano-Fujii M
    6. Sasayama M
    7. Utsumi T
    8. Hotta H

    (2008) Novel subgenotypes of hepatitis B virus genotypes C and D in papua, Indonesia

    Journal of Clinical Microbiology 46:2160–2166.

    https://doi.org/10.1128/JCM.01681-07

    • Google Scholar
39. Website

    1. Maddison WP
    2. Maddison DR

    (2017) Mesquite: a modular system for evolutionary analysis

    Version 3.2 Ed.

    http://mesquiteproject.org
40. 1. Makuwa M
    2. Souquière S
    3. Telfer P
    4. Apetrei C
    5. Vray M
    6. Bedjabaga I
    7. Mouinga-Ondeme A
    8. Onanga R
    9. Marx PA
    10. Kazanji M
    11. Roques P
    12. Simon F

    (2006) Identification of hepatitis B virus subgenotype A3 in rural Gabon

    Journal of Medical Virology 78:1175–1184.

    https://doi.org/10.1002/jmv.20678

    • PubMed
    • Google Scholar
41. 1. Martin DP
    2. Murrell B
    3. Golden M
    4. Khoosal A
    5. Muhire B

    (2015) RDP4: detection and analysis of recombination patterns in virus genomes

    Virus Evolution 1:vev003.

    https://doi.org/10.1093/ve/vev003

    • PubMed
    • Google Scholar
42. 1. McMahon BJ

    (2009) The influence of hepatitis B virus genotype and subgenotype on the natural history of chronic hepatitis B

    Hepatology International 3:334–342.

    https://doi.org/10.1007/s12072-008-9112-z

    • PubMed
    • Google Scholar
43. 1. Meldal BH
    2. Moula NM
    3. Barnes IH
    4. Boukef K
    5. Allain JP

    (2009) A novel hepatitis B virus subgenotype, D7, in tunisian blood donors

    Journal of General Virology 90:1622–1628.

    https://doi.org/10.1099/vir.0.009738-0

    • PubMed
    • Google Scholar
44. 1. Mühlemann B
    2. Jones TC
    3. Damgaard PB
    4. Allentoft ME
    5. Shevnina I
    6. Logvin A
    7. Usmanova E
    8. Panyushkina IP
    9. Boldgiv B
    10. Bazartseren T
    11. Tashbaeva K
    12. Merz V
    13. Lau N
    14. Smrčka V
    15. Voyakin D
    16. Kitov E
    17. Epimakhov A
    18. Pokutta D
    19. Vicze M
    20. Price TD
    21. Moiseyev V
    22. Hansen AJ
    23. Orlando L
    24. Rasmussen S
    25. Sikora M
    26. Vinner L
    27. Osterhaus A
    28. Smith DJ
    29. Glebe D
    30. Fouchier RAM
    31. Drosten C
    32. Sjögren KG
    33. Kristiansen K
    34. Willerslev E

    (2018) Ancient hepatitis B viruses from the bronze age to the medieval period

    Nature 557:418–423.

    https://doi.org/10.1038/s41586-018-0097-z

    • PubMed
    • Google Scholar
45. 1. Norder H
    2. Couroucé AM
    3. Coursaget P
    4. Echevarria JM
    5. Lee SD
    6. Mushahwar IK
    7. Robertson BH
    8. Locarnini S
    9. Magnius LO

    (2004) Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HBsAg subtypes

    Intervirology 47:289–309.

    https://doi.org/10.1159/000080872

    • PubMed
    • Google Scholar
46. 1. Olinger CM
    2. Venard V
    3. Njayou M
    4. Oyefolu AO
    5. Maïga I
    6. Kemp AJ
    7. Omilabu SA
    8. le Faou A
    9. Muller CP

    (2006) Phylogenetic analysis of the precore/core gene of hepatitis B virus genotypes E and A in West Africa: new subtypes, mixed infections and recombinations

    Journal of General Virology 87:1163–1173.

    https://doi.org/10.1099/vir.0.81614-0

    • PubMed
    • Google Scholar
47. 1. Osiowy C
    2. Larke B
    3. Giles E

    (2011) Distinct geographical and demographic distribution of hepatitis B virus genotypes in the Canadian Arctic as revealed through an extensive molecular epidemiological survey

    Journal of Viral Hepatitis 18:e11–e19.

    https://doi.org/10.1111/j.1365-2893.2010.01356.x

    • PubMed
    • Google Scholar
48. 1. Paraskevis D
    2. Angelis K
    3. Magiorkinis G
    4. Kostaki E
    5. Ho SY
    6. Hatzakis A

    (2015) Dating the origin of hepatitis B virus reveals higher substitution rate and adaptation on the branch leading to F/H genotypes

    Molecular Phylogenetics and Evolution 93:44–54.

    https://doi.org/10.1016/j.ympev.2015.07.010

    • PubMed
    • Google Scholar
49. 1. Paraskevis D
    2. Magiorkinis G
    3. Magiorkinis E
    4. Ho SY
    5. Belshaw R
    6. Allain JP
    7. Hatzakis A

    (2013) Dating the origin and dispersal of hepatitis B virus infection in humans and primates

    Hepatology 57:908–916.

    https://doi.org/10.1002/hep.26079

    • PubMed
    • Google Scholar
50. 1. Parekh S
    2. Zoulim F
    3. Ahn SH
    4. Tsai A
    5. Li J
    6. Kawai S
    7. Khan N
    8. Trépo C
    9. Wands J
    10. Tong S

    (2003) Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants

    Journal of Virology 77:6601–6612.

    https://doi.org/10.1128/JVI.77.12.6601-6612.2003

    • PubMed
    • Google Scholar
51. 1. Patterson Ross Z
    2. Klunk J
    3. Fornaciari G
    4. Giuffra V
    5. Duchêne S
    6. Duggan AT
    7. Poinar D
    8. Douglas MW
    9. Eden JS
    10. Holmes EC
    11. Poinar HN

    (2018) The paradox of HBV evolution as revealed from a 16th century mummy

    PLoS Pathogens 14:e1006750.

    https://doi.org/10.1371/journal.ppat.1006750

    • PubMed
    • Google Scholar
52. 1. Petzold DR
    2. Tautz B
    3. Wolf F
    4. Drescher J

    (1999) Infection chains and evolution rates of hepatitis B virus in cardiac transplant recipients infected nosocomially

    Journal of Medical Virology 58:1–10.

    https://doi.org/10.1002/(SICI)1096-9071(199905)58:1<1::AID-JMV1>3.0.CO;2-M

    • PubMed
    • Google Scholar
53. 1. Pourkarim MR
    2. Amini-Bavil-Olyaee S
    3. Kurbanov F
    4. Van Ranst M
    5. Tacke F

    (2014) Molecular identification of hepatitis B virus genotypes/subgenotypes: revised classification hurdles and updated resolutions

    World Journal of Gastroenterology 20:7152–7168.

    https://doi.org/10.3748/wjg.v20.i23.7152

    • PubMed
    • Google Scholar
54. 1. Pourkarim MR
    2. Amini-Bavil-Olyaee S
    3. Lemey P
    4. Maes P
    5. Van Ranst M

    (2010) Are hepatitis B virus "subgenotypes" defined accurately?

    Journal of Clinical Virology 47:356–360.

    https://doi.org/10.1016/j.jcv.2010.01.015

    • PubMed
    • Google Scholar
55. 1. Pourkarim MR
    2. Verbeeck J
    3. Rahman M
    4. Amini-Bavil-Olyaee S
    5. Forier AM
    6. Lemey P
    7. Maes P
    8. Van Ranst M

    (2009) Phylogenetic analysis of hepatitis B virus full-length genomes reveals evidence for a large nosocomial outbreak in Belgium

    Journal of Clinical Virology 46:61–68.

    https://doi.org/10.1016/j.jcv.2009.06.015

    • PubMed
    • Google Scholar
56. 1. Price MN
    2. Dehal PS
    3. Arkin AP

    (2010) FastTree 2--approximately maximum-likelihood trees for large alignments

    PLoS One 5:e9490.

    https://doi.org/10.1371/journal.pone.0009490

    • PubMed
    • Google Scholar
57. 1. Schaefer S

    (2007) Hepatitis B virus genotypes in Europe

    Hepatology Research 37:S20–S26.

    https://doi.org/10.1111/j.1872-034X.2007.00099.x

    • PubMed
    • Google Scholar
58. 1. Schweitzer A
    2. Horn J
    3. Mikolajczyk RT
    4. Krause G
    5. Ott JJ

    (2015) Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013

    The Lancet 386:1546–1555.

    https://doi.org/10.1016/S0140-6736(15)61412-X

    • Google Scholar
59. 1. Simmonds P
    2. Midgley S

    (2005) Recombination in the genesis and evolution of hepatitis B virus genotypes

    Journal of Virology 79:15467–15476.

    https://doi.org/10.1128/JVI.79.24.15467-15476.2005

    • PubMed
    • Google Scholar
60. 1. Stamatakis A

    (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies

    Bioinformatics 30:1312–1313.

    https://doi.org/10.1093/bioinformatics/btu033

    • PubMed
    • Google Scholar
61. 1. Sugauchi F
    2. Kumada H
    3. Acharya SA
    4. Shrestha SM
    5. Gamutan MT
    6. Khan M
    7. Gish RG
    8. Tanaka Y
    9. Kato T
    10. Orito E
    11. Ueda R
    12. Miyakawa Y
    13. Mizokami M

    (2004) Epidemiological and sequence differences between two subtypes (Ae and Aa) of hepatitis B virus genotype A

    Journal of General Virology 85:811–820.

    https://doi.org/10.1099/vir.0.79811-0

    • PubMed
    • Google Scholar
62. 1. Tallo T
    2. Tefanova V
    3. Priimägi L
    4. Schmidt J
    5. Katargina O
    6. Michailov M
    7. Mukomolov S
    8. Magnius L
    9. Norder H

    (2008) D2: major subgenotype of hepatitis B virus in Russia and the Baltic region

    Journal of General Virology 89:1829–1839.

    https://doi.org/10.1099/vir.0.83660-0

    • PubMed
    • Google Scholar
63. 1. Tanaka Y
    2. Hasegawa I
    3. Kato T
    4. Orito E
    5. Hirashima N
    6. Acharya SK
    7. Gish RG
    8. Kramvis A
    9. Kew MC
    10. Yoshihara N
    11. Shrestha SM
    12. Khan M
    13. Miyakawa Y
    14. Mizokami M

    (2004) A case-control study for differences among hepatitis B virus infections of genotypes A (subtypes Aa and Ae) and D

    Hepatology 40:747–755.

    https://doi.org/10.1002/hep.20365

    • PubMed
    • Google Scholar
64. 1. Tatematsu K
    2. Tanaka Y
    3. Kurbanov F
    4. Sugauchi F
    5. Mano S
    6. Maeshiro T
    7. Nakayoshi T
    8. Wakuta M
    9. Miyakawa Y
    10. Mizokami M

    (2009) A genetic variant of hepatitis B virus divergent from known human and ape genotypes isolated from a Japanese patient and provisionally assigned to new genotype J

    Journal of Virology 83:10538–10547.

    https://doi.org/10.1128/JVI.00462-09

    • PubMed
    • Google Scholar
65. 1. Thai H
    2. Campo DS
    3. Lara J
    4. Dimitrova Z
    5. Ramachandran S
    6. Xia G
    7. Ganova-Raeva L
    8. Teo CG
    9. Lok A
    10. Khudyakov Y

    (2012) Convergence and coevolution of hepatitis B virus drug resistance

    Nature Communications 3:789.

    https://doi.org/10.1038/ncomms1794

    • PubMed
    • Google Scholar
66. 1. Thedja MD
    2. Muljono DH
    3. Nurainy N
    4. Sukowati CH
    5. Verhoef J
    6. Marzuki S

    (2011) Ethnogeographical structure of hepatitis B virus genotype distribution in Indonesia and discovery of a new subgenotype, B9

    Archives of Virology 156:855–868.

    https://doi.org/10.1007/s00705-011-0926-y

    • PubMed
    • Google Scholar
67. 1. Yousif M
    2. Kramvis A

    (2013) Genotype D of hepatitis B virus and its subgenotypes: an update

    Hepatology Research 43:355–364.

    https://doi.org/10.1111/j.1872-034X.2012.01090.x

    • PubMed
    • Google Scholar
68. 1. Zehender G
    2. Ebranati E
    3. Gabanelli E
    4. Shkjezi R
    5. Lai A
    6. Sorrentino C
    7. Lo Presti A
    8. Basho M
    9. Bruno R
    10. Tanzi E
    11. Bino S
    12. Ciccozzi M
    13. Galli M

    (2012) Spatial and temporal dynamics of hepatitis B virus D genotype in Europe and the Mediterranean Basin

    PLoS One 7:e37198.

    https://doi.org/10.1371/journal.pone.0037198

    • PubMed
    • Google Scholar
69. 1. Zehender G
    2. Ebranati E
    3. Gabanelli E
    4. Sorrentino C
    5. Lo Presti A
    6. Tanzi E
    7. Ciccozzi M
    8. Galli M

    (2014) Enigmatic origin of hepatitis B virus: an ancient travelling companion or a recent encounter?

    World Journal of Gastroenterology 20:7622–7634.

    https://doi.org/10.3748/wjg.v20.i24.7622

    • PubMed
    • Google Scholar

Thank you for submitting your article "Unravelling the epidemic history of hepatitis B virus genotypes A and D using a full-genome phylogeographic approach" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Carla Osiowy as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Gisela Storz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Federico García (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The manuscript describes an analysis of global phylogeny and regional clustering of HBV genotypes A and D based on full genome, non-recombinant sequences available and downloaded from public sequence databases. Using phylogenetic methods, they have examined the clustering for these sequences to identify regional differences, the putative geographic origin and global dissemination of HBV A and D subgenotypes. Genotype A and D putatively originated in sub-Saharan Africa (Abstract, and Discussion, second and fifth paragraphs) and the region of North Africa/Middle East, respectively, and dispersal resulted in regional prevalence of specific sub-genotypes observed today. The article is well written and very interesting, particularly within the field of HBV and viral evolution, but would hopefully be of interest to evolutionary biologists, archaeologists, etc., due to the unique origin and evolutionary features of HBV serving as a complementary anthropological tool. In general the conclusions are reasonable from the results presented. There are only a few comments or concerns regarding aspects that require correction and regarding the methodology and result interpretation.

Essential revisions:

1) Study results regarding regional clustering and putative genotype origin are based on the geographic location as provided by submissions to public sequence databases. How reliable is this information? In particular, how did you control for database submissions in which the researcher's country of residence (or location of research) was listed as opposed to the patient's region of birth or residence prior to immigration (if relevant)? Referring to the Materials and methods subsection “DNA sequences, alignment and HBV genotyping”, the authors have downloaded all NCBI sequences for Genotypes A and D, then analysed these with a reference set of 110 sequences downloaded from NCBI. Did they remove any duplicated sequences from these 2 datasets? The authors also need to explain how duplicated sequences were identified and removed from the dual datasets from NCBI and HBVdb.

2) Further to comment 1, there is some concern regarding potential sampling bias. Were sequences from heterochronous sampling included in the analysis? Multiple sequences from single patients may skew prevalence estimates and thus overestimate regional clustering and more important, assumptions of monophyly. Similarly, database submissions related to a local common source transmission/outbreak may also overestimate monophyletic regional clustering. Was validation of the database selection performed to control for this? Some discussion is warranted regarding the potential limitation and bias that may be introduced.

3) The phylogeographic analysis performed did not result in molecular clock or temporal properties of the sequences. Yet inference is made regarding dispersal among migrating populations over time in the Discussion. How was this reconciled with the data? The authors had provided a message to reviewers regarding a new article describing the detection and analysis of HBV in ancient human bone and teeth samples. Going forward, it will be important to refer or discuss these findings in light of the present study results. For example, Mühlemann et al. and another recent paper (Krause-Kyora et al., 2018, eLife, ahead of print May 10 "Neolithic and Medieval virus genomes reveal complex evolution of Hepatitis B") describe HBV genotypes existing in locations inconsistent with present day distribution. Similarly, extinct lineages suggest a level of diversity that is no longer observable when estimating past origin and dispersal patterns using modern samples. Thus these new insights from the recent articles could be discussed within the manuscript and interpretation of results clarified.

4) It would be preferable to use the most current, validated naming system for all HBV subgenotypes throughout the manuscript to support appropriate naming/classification and avoid confusion for the reader. This has the benefit of reducing the number of supplementary figures (as well, Figures 1A and 2A and Supplementary Figures 1A and 2A appear to be almost identical – the supplementary figures are not needed). For example, in the Introduction, the sub-genotype numbering for genotype D needs to be better explained with regard to the previous and current sub-genotype designations. This has been explained well for genotype A in the next paragraph where the "former" sub-genotypes are also indicated, but this needs to be included for the genotype D paragraph for clarity. In the Materials and methods subsection “HBV nomenclature”, the authors state that they present their results using both the earlier and updated nomenclature, but have not done this for genotype D sub-genotypes. It may be easier to comprehensively explain the earlier and updated nomenclature in the Introduction, or Materials and methods, and then use the updated nomenclature throughout the rest of the paper, included the figures.

5) Materials and methods subsection “Recombination analysis and country grouping”. The authors indicate their finding that sub-genotype D5 was recombinant. This has not been previously noted and requires explanation or discussion. Also, sub-genotype D8 was not found to be recombinant, although this sequence has previously been noted as a recombinant sequence. This also needs an explanation.

6) Figure 1B needs correction with regard to the sub-genotype D4 sequences. According to Figure 1A, the only D4 sequences are from Latin America, the Caribbean, Australasia and Oceania. However, the coloured circles in Figure 1B indicate that sub-genotype sequences for D4 are also found in South Asia, South East Asia, East Asia and North America. Where are the sequences to explain this figure?

7) Figure 2B indicates the putative origin of genotype A is Middle East/Central Asia, as shown similarly to Figure 1B for genotype D, however the comments in the text suggest the origin was in Sub-Saharan Africa, and the figure legend indicates the exact geographic origin is unknown. The figure should be modified to reflect this.

Essential revisions:

1) Study results regarding regional clustering and putative genotype origin are based on the geographic location as provided by submissions to public sequence databases. How reliable is this information? In particular, how did you control for database submissions in which the researcher's country of residence (or location of research) was listed as opposed to the patient's region of birth or residence prior to immigration (if relevant)? Referring to the Materials and methods subsection “DNA sequences, alignment and HBV genotyping”, the authors have downloaded all NCBI sequences for Genotypes A and D, then analysed these with a reference set of 110 sequences downloaded from NCBI. Did they remove any duplicated sequences from these 2 datasets? The authors also need to explain how duplicated sequences were identified and removed from the dual datasets from NCBI and HBVdb.

We went through all published studies reporting HBV sequences used in the analysis (Supplementary files 3 and 4). Detailed information about the country of birth of the HBV infected patients was not available. The following sentence was included in the Materials and methods: “Detailed information about the country of birth or the immigration status of patients for whom HBV sequences included in the analysis was not available”. The lack of information about the country of birth or the immigration status of patients for whom HBV sequences was mentioned as a potential limitation of our study.

Duplicate sequences from NCBI and HBVdb were removed if they had the same accession number.

The previous sentence was included in the Materials and methods.

2) Further to comment 1, there is some concern regarding potential sampling bias. Were sequences from heterochronous sampling included in the analysis? Multiple sequences from single patients may skew prevalence estimates and thus overestimate regional clustering and more important, assumptions of monophyly. Similarly, database submissions related to a local common source transmission/outbreak may also overestimate monophyletic regional clustering. Was validation of the database selection performed to control for this? Some discussion is warranted regarding the potential limitation and bias that may be introduced.

We checked all published papers for the presence of an “outbreak”. This was reported for three studies of genotype D, one from Germany (N=1 sequence; PMID=10223539), one from the USA (N=7 sequences, PMID: 15382123) and one from India (N=39, PMID 21108697) (Supplementary file 3). The outbreak sequences corresponded to 5.1% (47 of 926) of the genotype D sequences. Similarly, for genotype A “outbreaks” were reported in two studies, one from USA (N=3 sequences, PMID: 12767980) and one from Belgium (N=58 sequences, PMID: 19615936) (Supplementary file 4), corresponding to 8.3% (61 of 731) of the genotype A sequences analysed in our study.

We also looked for the presence of multiple sequences from individual patients and we found: i) for two cases (N=7 clones; country of sampling: India) and (N=5 clones; country of sampling: Italy) multiple sequences were available for genotype D (data available on NCBI but not published in scientific journal). We also found for two studies: i) (N=238 multiple sequences from 4 patients; country of sampling USA; PMID: 22510694) and ii) (N=5 multiple sequences; country of sampling Germany; PMID: 15962285) that multiple sequences were available for genotype A.

The previous text was included in Materials and methods. We repeated the whole analysis for both genotypes and we revised the text and the figures.

3) The phylogeographic analysis performed did not result in molecular clock or temporal properties of the sequences. Yet inference is made regarding dispersal among migrating populations over time in the Discussion. How was this reconciled with the data? The authors had provided a message to reviewers regarding a new article describing the detection and analysis of HBV in ancient human bone and teeth samples. Going forward, it will be important to refer or discuss these findings in light of the present study results. For example, Mühlemann et al. and another recent paper (Krause-Kyora et al., 2018, eLife, ahead of print May 10 "Neolithic and Medieval virus genomes reveal complex evolution of Hepatitis B") describe HBV genotypes existing in locations inconsistent with present day distribution. Similarly, extinct lineages suggest a level of diversity that is no longer observable when estimating past origin and dispersal patterns using modern samples. Thus these new insights from the recent articles could be discussed within the manuscript and interpretation of results clarified.

We discussed the new findings about the analysis of HBV from ancient samples from both papers in the discussion. We also discuss the importance of these findings with regard to our findings and the inference of the putative origin of genotypes A and D.

4) It would be preferable to use the most current, validated naming system for all HBV subgenotypes throughout the manuscript to support appropriate naming/classification and avoid confusion for the reader. This has the benefit of reducing the number of supplementary figures (as well, Figures 1A and 2A and Supplementary Figures 1A and 2A appear to be almost identical – the supplementary figures are not needed). For example, in the Introduction, the sub-genotype numbering for genotype D needs to be better explained with regard to the previous and current sub-genotype designations. This has been explained well for genotype A in the next paragraph where the "former" sub-genotypes are also indicated, but this needs to be included for the genotype D paragraph for clarity. In the Materials and methods subsection “HBV nomenclature”, the authors state that they present their results using both the earlier and updated nomenclature, but have not done this for genotype D sub-genotypes. It may be easier to comprehensively explain the earlier and updated nomenclature in the Introduction, or Materials and methods, and then use the updated nomenclature throughout the rest of the paper, included the figures.

We revised the text accordingly and we introduced the most current, validated naming system for all HBV subgenotypes. The number of supplementary figures has been reduced. We included a section in “Phylogenetic and phylogeographic analysis” paragraph about the revised nomenclature and the extensive recombination analysis performed for all putative recombinant subgenotypes of D. We found that there is no evidence for recombination for D4, D6, D7 and D8 subgenotypes.

5) Materials and methods subsection “Recombination analysis and country grouping”. The authors indicate their finding that sub-genotype D5 was recombinant. This has not been previously noted and requires explanation or discussion. Also, sub-genotype D8 was not found to be recombinant, although this sequence has previously been noted as a recombinant sequence. This also needs an explanation.

Recombination analysis using bootscanning and RDP4 for RS-D5 showed that several RS-D5 sequences were intragenotypic recombinants consisting of diverse mosaic patterns. Phylogenetic analysis of the two subgenomes of the full-length HBV genome (1-2000 and 2001-3078 nts; corresponding to sites 60-2059, 2060-3179 of the reference X02496) revealed discordant phylogenetic signal with regard to the clustering of RS-D5. Specifically, in the first part of the genome (1-2000 nts) RS-D5 clustered as an outlier to genotype D, while in the second half of the genome (2001-3078 nts) RS-D5 clustered within genotype D. The discordant clustering of the RS-D5 prompted us to perform phylogenetic analysis with and without RS-D5. We also report the results of the phylogeographic analysis and the dispersal pathways of genotype D after the inclusion of RS-D5 in the results and in separate figures (Figure 1—figure supplement 2, Figure 1—figure supplement 3, Figure 2—figure supplement 1)

6) Figure 1B needs correction with regard to the sub-genotype D4 sequences. According to Figure 1A, the only D4 sequences are from Latin America, the Caribbean, Australasia and Oceania. However, the coloured circles in Figure 1B indicate that sub-genotype sequences for D4 are also found in South Asia, South East Asia, East Asia and North America. Where are the sequences to explain this figure?

Figure 1B was revised (it now appears as Figure 2). We analysed D4 sequences from Latin America (Brazil), the Caribbean (Martinique, Haiti), Australasia (Australia, New Zealand), Oceania (Tonga, Kiribati, Fiji, Papua New Guinea, Samoa) and North America (Canada). Sequences from North America (Canada) clustered within the green LTN from the Caribbean (Martinique, Haiti) presented in Figure 1.

7) Figure 2B indicates the putative origin of genotype A is Middle East/Central Asia, as shown similarly to Figure 1B for genotype D, however the comments in the text suggest the origin was in Sub-Saharan Africa, and the figure legend indicates the exact geographic origin is unknown. The figure should be modified to reflect this.

We revised the text accordingly and the figure legend (Figure 2B appears now as Figure 4). We introduced the following sentence in the results to explain why the putative origin of genotype A was in Middle East/Central Asia: “The separate phylogenetic branching of clades I and II and the rest of genotype A sequences together with the fact that they have spread into two different geographic areas suggest that the putative origin of genotype A is close to the Africa and Europe and probably in Middle East/Central Asia (Figure 4)”.

Author details

1. Evangelia-Georgia Kostaki

   Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, Athens, Greece

   Contribution

   Formal analysis, Investigation, Writing—original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3346-0930

2. Timokratis Karamitros

   1. Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, Athens, Greece
   2. Department of Zoology, University of Oxford, Oxford, United Kingdom

   Contribution

   Formal analysis, Investigation, Writing—original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0841-9159

3. Garyfallia Stefanou

   Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, Athens, Greece

   Contribution

   Data curation, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

4. Ioannis Mamais

   Department of Health Sciences, School of Sciences, European University of Cyprus, Nicosia, Cyprus

   Contribution

   Data curation, Formal analysis, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

5. Konstantinos Angelis

   Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, Athens, Greece

   Contribution

   Data curation, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

6. Angelos Hatzakis

   Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, Athens, Greece

   Contribution

   Funding acquisition, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

7. Anna Kramvis

   Hepatitis Virus Diversity Research Unit, Department of Internal Medicine, Faculty of Health Science, University of the Witwatersrand, Johannesburg, South Africa

   Contribution

   Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

8. Dimitrios Paraskevis

   Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, Athens, Greece

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft

   For correspondence

   dparask@med.uoa.gr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6167-7152

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