Program Schedule

1224
Defining herpes simplex virus type 2 sequence heterogeneity using next generation sequencing

Session: Oral Abstract Session: Viral Infections: Clinical Trials and Pathogenesis
Friday, October 10, 2014: 2:30 PM
Room: The Pennsylvania Convention Center: 105-AB
Background:

Vaccines are most successful for pathogens in which wild-type infection leads to protection against homologous re-infection. For HSV-2, we have little information concerning natural protection from re-infection or the variability between strains that could modulate protection from challenge.  Understanding differences in HSV-2 strains worldwide, and the potential for reinfection, is essential in development of an HSV-2 vaccine.

Methods:

DNA from genital HSV-2 lesions containing ≥7 log10 copies HSV DNA/swab were sequenced using the Illumina next generation sequencing (NGS) platform.  Human and highly repetitive sequences were subtracted, and remaining reads were aligned to the reference HSV-2 genome.  Prevalent SNPs were defined as present in >10% and <90% of the sequenced strains. Informative SNPs were identified using FastTagger.  Bayesian computations were used to estimate probabilities of identifying super-infection.

Results:

Fifty-six samples from genital herpes lesions were sequenced.  Of 49 sequences from 39 persons with adequate coverage, the HSV genome had a median of 73.9 fold coverage (range 9.8-771 fold).  Sixteen persons (41%) were from Africa, 12 (31%) from Peru, and 11 (28%) from the US; one-third were HIV infected.  Of 2854 loci with heterogeneity, prevalent SNPs were found at 456 (16%) loci.  Using genotyping at the 6 most informative SNPs to distinguish strains, we estimate a 90% probability of correctly identifying super-infection or lack thereof.  Of 10 persons with paired specimens collected a median of 2.1 years apart, 2 (20%) had super-infection; both were HIV-infected.  In each case, the strains differed at 186 loci.  Six of 10 remaining pairs differed at 0 loci, and 2 differed at only 1 locus, indicating minimal within host evolution and low sequencing error.  

Conclusion:

High quality HSV-2 sequence data can be obtained directly from genital swabs, removing the need for culture which may introduce mutations.  The initial phase of our study has identified super-infection in 20% of persons investigated.  Further research will determine whether host immune status, gender, and sexual exposure history are correlates of super-infection.

Christine Johnston, MD, MPH1, Amalia Magaret, PhD2, Kurt Diem3, Matthew Fitzgibbon4, Meei-Li Huang, PhD3, Stacy Selke, MS3, Anna Rashevsky3, Jairam Lingappa, MD, PhD5, Connie Celum, MD, MPH6, David M. Koelle, MD1 and Anna Wald, MD, MPH, FIDSA7, (1)Medicine, University of Washington, Fred Hutchinson Cancer Research Center, Seattle, WA, (2)Department of Laboratory Medicine, University of Washington, Seattle, WA, (3)Laboratory Medicine, University of Washington, Seattle, WA, (4)Fred Hutchinson Cancer Research Center, Seattle, WA, (5)Global Health & Medicine, Pediatrics, Partners in Prevention, University of Washington, Seattle, WA, (6)Global Health & Medicine, University of Washington, Seattle, WA, (7)Department of Medicine, University of Washington, Seattle, WA

Disclosures:

C. Johnston, AiCuris: Investigator, Research support
Genocea: Investigator, Research support
Agenus: Investigator, Research support

A. Magaret, None

K. Diem, None

M. Fitzgibbon, None

M. L. Huang, None

S. Selke, None

A. Rashevsky, None

J. Lingappa, None

C. Celum, None

D. M. Koelle, Sanofi Pasteur: Research Contractor, Research support

A. Wald, Aicuris: Consultant, Consulting fee
Genocea: Investigator, Research support
Agenus: Investigator, Research support
Vical: Investigator, Research support

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