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

2. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
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,

3. Epidemiologic Features
Hepatitis E -
Epidemiologic Features
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.
UWI, St. Augustine, Trinidad & Tobago, 29 29 29

4. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Hepatitis E - Clinical Features
Incubation period: Average 40 days
Range days
Case-fatality rate: Overall, 1%-3% Pregnant women, 15%-25%
Illness severity: Increased with age
Chronic sequelae: None identified
UWI, St. Augustine, Trinidad & Tobago, 27 27 27

5. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.

6. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Hepatitis E Virus Genome
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,

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

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

11. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Blastp Protein ID from initial selected isolate
>gb|ACD | RNA-dependent RNA polymerase [Hepatitis E virus] Length=116
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
ClustalX2 multiple alignment
Analysis in Phylogenetic software
UWI, St. Augustine, Trinidad & Tobago,

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

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

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

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

16. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Phylogenetic tree: Phylip with DNA data
JAP291
Cub25_08
Mex017
Fin969
Fin973
Ind
LonInd097
Mor494
Chn001
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
UWI, St. Augustine, Trinidad & Tobago,

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

18. DNA Alignment without Cub25-2008
UWI, St. Augustine, Trinidad & Tobago,

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

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

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

22. UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
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,

23. Thank you for your attention!
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
UWI, St. Augustine, Trinidad & Tobago,