Transcript Phylogenetics

Phylogenetic relationships amongst
HEV strains
Participants:
•Agnes Zotter
•Lambert Motilal
•Maria Montalvo
•Marissa Moses
•Robin Antoine
Hepatitis E Virus
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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.
Hepatitis E Epidemiologic Features
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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, 29.01.2010.
Hepatitis E - Clinical Features
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Average 40 days
Range 15-60 days
Case-fatality rate:
Overall, 1%-3%
Pregnant women, 15%-25%
Illness severity:
Increased with age
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Chronic sequelae:
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Incubation period:
None identified
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
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, 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, 29.01.2010.
Methods:
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
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Blastp Protein ID from initial selected isolate
>gb|ACD39863.1| 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, 29.01.2010.
Protein alignment (MSA):
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Results:
R
Nucleic Acids Research 2005 33(Web Server Issue):W553W556; 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/
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, 29.01.2010.
Protein
Seq.
data
Phylogenetic tree
NJ
analysis
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Phylogenetic tree: Phylip with DNA data
JAP291
Cub25_08
Mex017
Fin969
Fin973
Ind
LonInd097
Mor494 Chn001
Hyder
Fin971
Fin967
US2
Chn363
Ind103
Chn457
Cub19_99
Cub9_99
Nind
Cub2_05
Cub27_99
Cub10_99
SKor476
US1
SKor466
Fr757
Fr719
Fr767
Fr787
Fr785
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Phylogenetic tree: Phylip/protein data
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
DNA Alignment without Cub25-2008
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Phylogenetic
tree:
MEGA4/DNA
data
Genotype 1
Genotype 2
Genotype 4
Genotype 3
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
Phylogenetic tree: MEGA/protein data
Genotype 1
Genotype 2
Genotype 4
Genotype 3
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
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Genotype 3 with American and European isolates
from swine and human
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
A take-home message…
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Avoid drinking water (and beverages with ice) of
unknown purity, uncooked shellfish, and
uncooked fruit/vegetables not peeled or prepared
by traveler.
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IG prepared from donors in Western countries
does not prevent infection.
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Unknown efficacy of IG prepared from donors in
endemic areas.
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Vaccine?
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.
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…
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Dr. Urmila Kulkarni-Kale
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Dr. Jessica Kissinger
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Dr. Dinesh Gupta
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Dr. Arnab Pain
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Dr. Vrijesh Tripathi
UWI, St. Augustine, Trinidad & Tobago, 29.01.2010.