Exploring Hepatitis B Virus Evolution and Impact on Human Health

1. ASTROBIOLOGY SCIENCE CONFERENCE 2006 Washington, DC March 25 – 31, 2006 Viral Evolution 03.29.06 HEPATITIS B VIRUS: COMMENTS ON VIRAL AND HUMAN EVOLUTION Baruch S. Blumberg Fox Chase Cancer Center Philadelphia, PA, 19111, USA

2. 1. Hepatitis B Virus is a very common virus. • 2. Humans have been infected with virus for a very long time, possibly from the time of the origin of humans. • 3. Viruses closely related to HBV (“Hepadna viruses”) are known in many species other than humans. • 4. HBV is a major deadly infectious agent. • 5. Males are more likely than females to become chronic carriers of HBV and to die as a consequence of infection. • 6. HBV related deaths are highest in older males; high • HBV prevalence decreases the male to female ratio in older age groups. • 7. There are a series of polymorphisms that affect susceptibility to chronic infection.

3. 8. Parents who are carriers of HBV have a higher ratio of males to females in their newborn offspring than do parents who have antibody against the surface antigen of HBV; high HBV incidence increases the male to female ratio at birth. • 9. There may be host advantages to the carrier state. • 10. The worldwide vaccination program is decreasing the prevalence of HBV. • 11. Virus sequences are very common in the human genome. • 12. HBV as an “engine” of human evolution. • 13. Were viruses an early form of life and, possibly, the earliest form?

4. Primary Cancer of the Liver • Worldwide: • Third most common cause of death from cancer in males • Seventh most common cause of death from cancer in females • More than a million deaths per year • Hepatitis B virus (about 85%) and hepatitis C virus account for most of these cancers • Many other factors involved in the pathogenesis

5. Human Hepatitis Viruses Genome Virus Genome Size (Kb) Envelope Classification HAV RNA 7.5 - Picornavirus (Hepatovirus) HBV DNA 3.2 HBsAg Hepadnavirus HCV RNA 9.4 + Pestivirus- or Flavivirus-like HDV RNA 1.7 HBsAg Unclassified HEV RNA 7.5 - Claicivirus-like or Alpha-like supergroup

6. HBV is one of the most common infections in the world. About 600,000 to 1,000,000 deaths per year are attributable to the diseases associated with HBV.

7. PREDICTED DEATHS FROM PREVALENT CARRIERS OF HIV AND HBV (H. Margolis, Center for Disease Control. Paper presented at ThirdInternational Conference on Therapies for Viral Hepatitis, Maui, 12/12/99) Total Deaths (Millions) Number Infected (Millions) Case Fatality Rate Virus HIV 34 100% 34 HBV 371 High 25% 92.8 Low 15% 55.7

8. “Hepatitis B is a viral infection of the liver. More than two thousand million (2 billion) people alive today have been infected with the hepatitis B virus. Approximately 350 million are chronically infected and are at high risk of serious illness and death from cirrhosis of the liverand primary liver cancer. Hepatitis B is preventable with a safe and effective vaccine — the first vaccine against cancer.” WHO website, 2004

10. MORTALITY BY AGE, GENDER AND HBV RESPONSE HAIMEN CITY, CHINA 2500 M HBV+ M HBV- F HBV+ F HBV- 2000 1500 Mortality per 100,000 py 1000 500 0 25-34 35-44 45-54 55-64 65-74 Age group (years) G. Chen, W. Lin, F. Shen, U. Iloeje, T. London and A. Evans Inter. J. of epid. 2004, in print

11. HEPATITIS B VIRUS MORPHOLOGY Characteristics Nucleic acid: DNA Classification:hepadnavirus type 1 Serotypes: multiple In vivo replication:reverse transcription inliver and other tissues In vitro propagation:primary hepatocyteculture and transfectionby cloned HBV DNA 22 nm 42 nm HBsAg C HBV DNA HBcAg

14. HEPATITIS B LIKE VIRUSES Hepadnaviruses Human Homo sapiens HBV Gorilla Gorilla sp. HBV Chimpanzee Pan troglodytes ChHBV Gibbon Family Hylobatidae HBV Orangutan Pongo pygmaeus HBV Woolly monkey Lagothrix lagotricha WMHBV Tree shrew Tupaia belangeri HBV (Kangaroo Family Macropodidae KHV) Woodchuck or Groundhog Marmota monax WHV Beechey ground squirrel Spermophilus beecheyi GSHV Arctic squirrel S. parryi kennicotti ASHBV Richardson ground squirrel S. richardsonii RGSHV (Tree squirrel Sciurus carolinensis THBV) Pekin duck Anas domesticus DHBV Great blue heron Ardea herodias HHBV Grey heron A. cinerea HHBV Snow goose Anser caerulescens SGHBV Ross’ goose A. rossii RGHBV White Stork Ciconia ciconia STHBV Demoiselle crane Anthropoides virgo CHBV Grey crowned crane Balearica regulorum CHBV (Stink snake Elaphe carinata SSHV) (Items in parentheses have not been validated. Tree shrews were experimentally infected.)

15. CONTROL OF HEPATITIS B VIRUS IN CHINA Z.Y. Xu, T.Q. Yan, S.J. Zhao, et al. Shanghai Medical University “Infant HBV immunization has been implemented into public health service in China since 1986… the seropositivity for HBsAg was reduced from 16.3% (879/5397) of historical controls to 1.43% (70/4886) in cross-sectional study and 1.81% (210/11582) in a cohort study.”

16. HBV VACCINATION IN THE UNITED STATES • In the United States, the number of new HBV infections per year has declined from an average of 260,000 in the 1980s to about 78,000 in 2001. The highest rate of disease occurs in the 20-49 age group. Greatest decline is the consequence of the routine HBV vaccination program among children and adolescents.  There are an estimated 1.25 million chronically infected Americans, of whom 20-30% acquired their infection in childhood. • CDC Website, 2004

17. HBV VACCINATION IN ALASKA, USA •  A universal childhood vaccination program was undertaken between 1981 and 1983 in Native Americans in Alaska, USA (McMahon, et al., 1996). •  Acute hepatitis B infection dropped from 215 cases/100,000 before the vaccination program, to 7 to 14 cases in 1993 after the program was in place. • In 1995, no cases were reported.

18. Hepatitis B Virus Worldwide Vaccination Program As of May 2003, 151 (79%) of 192 national members of the WHO had universal childhood vaccination programs. There are 89 member states that have been designated as having a high prevalence of HBV carriers. Sixty four (72%) have universal infant vaccination programs. It is the goal of the WHO to have vaccination programs in all countries by 2007.

19. Distribution of Australia Antigen (HBsAg) by Gender Total Number 243 226 495 430 334 764 138 59 197 45 44 89 Number Positive 19 14 33 27 10 37 6 3 9 10 6 16 Percent Positive 7.8 6.2 6.7 6.3 3.0 4.8 4.3 5.1 4.6 22.2 13.6 18.0 Marshall Islands, USTTPI Male Female Total Cebu, Philippines Male Female Total Manila, Philippines Male Female Total Cashinahua, Peru Male Female Total Blumberg, et al, Amer. J. Human Genetics 18, 594, 1966

20. PLATI, GREECE. NUMBER OF MALE AND FEMALE LIVE BIRTHS ACCORDING TO THE RESPONSES TO HBV OF PARENTS Sex ratio 250 (161,429)* 146 (96,230)* 109 (91,131)* Live births Male Females Parent’s response To HBV Either parent HBsAg + : anti-HBs – Both parents HBsAg - : anti-HBs – Both parents HBsAg - : either parent anti-HBs + Couples (No.) 33 29 154 60 (1.8 ± 0.2) 51 (1.8 ± 0.2) 24 (1.6 ± 0.1) 24 (0.7 ± 0.1) 35 (1.2 ± 0.2) 22 (1.4 ± 0.1) *In parentheses, the 5 percent confidence limits. Blumberg, B.S. Sex differences in response to Hepatitis B Virus, Arthritis and Rheumatism,22, 1261, 1979

21. Hepatitis B and Sex Ratio: Individual Level Estimates Daughters Location Greenland Greenland Kar Kar Island Kar Kar Island Greece 1 Greece 1 Philippines Philippines Greece 2 Greece 2 France France HBV Status Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Sons 64 174 63 163 85 287 66 304 52 1006 20 149 60 194 54 206 46 255 41 301 30 955 12 122 Sex Ratio 1.07 0.90 1.17 0.79 1.85 1.13 1.61 1.01 1.73 1.05 1.66 1.22 Notes: This table shows sex ratios among the children of carrier and non-carrier parents in four regions. Data were collected by testing married women and, in all cases except for Greenland, their husbands for HBV. Detailed reproductive histories were also collected. The table represents all births to women in those samples, with generally more than one birth to each women. The last two studies (Greece 2 and France) were designed specifically to test the hypothesis that HBV affects offspring sex ratio, and were run after the original theory was expressed. From Oster, E. 2004

22. SEX RATIO AND HEPATITIS, WORLD China 1.14 1.12 South Korea 1.1 Pakistan Belarus 1.08 Sex Ratio at Birth Spain Singapore Greece Ireland Turkey Bangladesh Poland Japan Malaysia Australia Israel 1.06 N. Amer & W. Europe Italy Brazil France 1.04 Mexico 1.02 Iran 1 0 2 4 6 8 10 12 14 16 Hepatitis Rate (%) Notes: Sex ratio is number of boys for each girl. Only countries with more than 15,000 people used to caclulate HBV pravalence are included. Citations for each country are in Appendix B. Oster, E., Hepatitis B and the Case of the Missing Women, Presentation, October 12, 2004

23. EFFECTS OF VACCINATION PROGRAM ALASKA • High HBV prevalence among Alaskan Native Americans; low among non-Native American • Universal vaccination program (including catch- up) instituted in early 1980s. Oster, E., Hepatitis B and the Case of the Missing Women, Presentation, October 12, 2004

24. CHANGES IN SEX RATIO IN ALASKA BEFORE AND DURING VACCINATION PROGRAM 1.16 Native American High HBV Native American Low HBV Non-Native American 1.14 1.12 1.1 1.08 1.06 1.04 1.02 1 1980-1985 1986-1990 1991-1995 1996-2002 Oster, E., Hepatitis B and the Case of the Missing Women, Presentation, October 12, 2004

25. • HBV explains about 50% of the missing women overall: over 90% in China, 11% in India • Vaccination programs appear to decrease sex ratio Oster, E., Hepatitis B and the Case of the Missing Women, Presentation, October 12, 2004

26. Microorganism Gene Affinity Clusters

27. Microorganism Gene Affinity Clusters (cont.)

28. HBV and Iron in Down’s Syndrome Patients With and Without HBsAg HBsAg(+) Mean SD HBsAg(+) Mean SD Hemoglobin g/100ml 15.2 1.4 14.9 1.4 Hematocrit % 44.2 3.8 43.5 3.6 Serum Iron mg/100ml 163.7 115.1 84.1 33.6 TIBC mg/100ml 250.4 108.2 356.8 144.8 Increased Hemoglobin, Hematocrit, Serum Iron and decreased Total Iron Binding Capacity (TIBC) in 20 Down’s Syndrome patients with serum HBsAg compared to 20 without. The differences are significant. Sutnick, Blumberg, Lustbader. Ann. Int. Med. 81, 855, 1974

29. Serum Iron Levels (mg/dl) Senegal Male Female 77 81 75 58 81 112 HBsAg (+) HBsAg (-) Number Downs Syndrome Male Female 164 - 84 - 40 - Total 164 84 40 Renal Dialysis Male Female 114 214 98 100 40 117 Total 145 100 220 Total 79 65 193 Serum Iron levels in 1) Patients with Down’s Syndrome 2) Patients on renal dialysis and 3) Residents of a rural community in Senegal, West Africa. Blumberg in Szentivanyi and Friedman, Editors, Viruses, Immunity and Immunodeficiency, Plenum 1986

30. Large numbers of retroviral sequences (including those of HBV) are found in the human genome. These are often the “fossil” record of viruses that were incorporated into the germ line in previous generations. It is estimated that 8% of the modern human genome is made of retroviral sequences, a larger number than the coding sequences, and they are often highly conserved. Modified from Blumberg, B. S. 2005, Hepatitis B virus. Conjectures on human interactions and the origin of life, in, “Life As We Know It”, Volume 10 of the series “Cellular Origins, Life in Extreme Habitats and Astrobiology” edited by J. Seckbach, Springer, Dordrecht, NL.; and Kilbourne, E. D. (2004). Virus paleontology, disease, and evolution: an introduction. Proc. Amer. Philosophical Society 148: 261 and following articles.

31. SUMMARY (1) • Hepatitis B vaccination is one of the largest worldwide disease prevention programs. • It has decreased the spread of HBV, particularly in China and East Asia. It has significantly decreased morbidity from liver disease and prevented the death of millions. • HBV vaccination appears to prevent primary cancer of the liver; it is the first widely used preventive cancer vaccine.

32. SUMMARY (2) • There are important non-pathological interactions of HBV with Humans. Parents who are carriers of HBV have a higher ratio of males to females among their offspring than parents who developed antibody against the surface antigen. This may account for the high sex ratios seen in China and in other areas with a high prevalence of HBV infection. The apparent “loss” of females in these populations may be ascribed, at least in part, to HBV infection. • In two studied populations the HBV vaccination has decreased the ratio of males to females among newborns. If confirmed, this may have important, biological, demographic and economic effects.

33. SUMMARY (3) • The response of the host to HBV infection is related to a series of polymorphic locii that are, in turn, related to other disease causing agents. • There may be advantages to the HBV carrier state at certain periods of the hosts life, i.e. there is increased iron retention in HBV carriers compared to those with anti-HBs and uninfected people. • HBV is integrated into the genomes of tens of millions of humans.