1. Phylogenetic relationships amongst HEV strains Participants: Agnes Zotter Lambert Motilal Maria Montalvo Marissa Moses Robin Antoine

2. Hepatitis E Virus Hepeviridae, Hepevirus genus Unenveloped RNA virus, 27-34nm in diameter +ve stranded RNA genome, 7.2 kb in size Very labile and sensitive UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.

3. Most outbreaks associated with faeceally contaminated drinking water. Large epidemics have occurred in the Indian subcontinent, USSR, China, Africa and Mexico. In the United States and other non-endemic areas, where there are no documented outbreaks of hepatitis E, a low prevalence of anti-HEV (<2%) has been found in healthy populations. The source of infection for these persons is unknown. Minimal person-to-person transmission. Hepatitis E - Epidemiologic Features UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.

4. Incubation period:Average 40 days Range 15-60 days Case-fatality rate:Overall, 1%-3% Pregnant women, 15%-25% Illness severity:Increased with age Chronic sequelae:None identified Hepatitis E - Clinical Features UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.

6. HEV isolates have been grouped into four genotypes (1 to 4) Genotype 1 groups isolates found mainly in Asia and Africa Genotype 2 contains an isolate from an outbreak in Mexico and Africa Genotype 3 groups isolates found in the US & Europe Genotype 4 groups isolates found in China, Taiwan and Japan UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Hepatitis E Virus Genome

7. Research question UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. To determine the genotype identity of isolates found in Cuba Possible ways of managing the disease

8. Methods: UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Sequences from RNA polymerase in GenBanK (NCBI) 13 hits CUB9-1999 isolate chosen (241 bp linear RNA) Blastn: 30 entries chosen/E-value Length +/- 10 nucleotides alignment

11. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Blastp Protein ID from initial selected isolate ClustalX2 multiple alignment Analysis in Phylogenetic software >gb|ACD39863.1| RNA-dependent RNA polymerase [Hepatitis E virus] Length=116gb|ACD39863.1| Score = 133 bits (334), Expect = 6e - 37, Method: Compositional matrix adjust. Identities = 61/79 (77%), Positives = 69/79 (87%), Gaps = 0/79 (0%) Query 1 CALFGPWFRAIEKAILALLPQGVFYGDAFDDTVFSATVAAAKASMVFENDFSEFDSTQNN 60 CALFGPWFRAIEK ILALLP +FYGDA++++VFSA ++ A +SMVFENDFSE ST NN Sbjct 9 CALFGPWFRAIEKE ILALLPPN IFYGDAYEES VFSAAISGAGSSMVFENDFSEDXSTLNN 68 Query 61 FSLGLECAIMEECGMPQWL 79 FSLGLEC IMEECGMPQWL Sbjct 69 FSLGLECVIMEECGMPQWL 87

12. Protein alignment (MSA): UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.

13. Results: UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. R Nucleic Acids Research 2005 33(Web Server Issue):W553- W556; doi:10.1093/nar/gki494. C-Y Lin *, F-K Lin, CH Lin, L-W Lai, H-J Hsu, S-H Chen and CA Hsiung http://power.nhri.org.tw /

14. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Genotype 1 Genotype 3 Genotype 4 Genotype 2 DNA data NJ analysis Phylogenetic tree:

15. Protein Seq. data NJ analysis UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Phylogenetic tree

16. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Phylogenetic tree: Phylip with DNA data JAP291 Cub25_08 Mex017 Fin969 Fin973 Ind LonInd097 Mor494Chn001 Chn363 Ind103 Chn457 Hyder Cub19_99 Cub9_99 Nind Cub2_05 Cub27_99 Cub10_99 Fin971 Fin967 US2 SKor476 US1 SKor466 Fr757 Fr719 Fr767 Fr787 Fr785

17. Phylogenetic tree: Phylip/protein data UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.

18. DNA Alignment without Cub25-2008

19. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Phylogenetic tree: MEGA4/DNA data Genotype 1 Genotype 2 Genotype 3 Genotype 4

20. Phylogenetic tree: MEGA/protein data UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Genotype 1 Genotype 2 Genotype 3 Genotype 4

21. HEV from human in Cuba were clustered in at least two genotypes. Conclusion UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Genotype 1 with Asian and African isolates Genotype 3 with American and European isolates from swine and human

22. A take-home message… Avoid drinking water (and beverages with ice) of unknown purity, uncooked shellfish, and uncooked fruit/vegetables not peeled or prepared by traveler. IG prepared from donors in Western countries does not prevent infection. Unknown efficacy of IG prepared from donors in endemic areas. Vaccine? UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.

23. Thank you for your attention! UWI, St. Augustine, Trinidad & Tobago, 29.01.2010. Special thanks for the great teachers, who were very patient and helpful, who helped us to make this presentation better… Dr. Urmila Kulkarni-Kale Dr. Jessica Kissinger Dr. Dinesh Gupta Dr. Arnab Pain Dr. Vrijesh Tripathi