NextGen Sequencing to Characterize Near Full Length Human Immuno

Deficiency virus (HIV-1) Subtypes Evolving Within Infected Host

Supraja Kanipakam, Viswanath Ragupathy Ph.D, Andrew Hwang and Indira Hewlett Ph.D
Laboratory of Molecular Virology, FDA/CBER/OBRR, Silver Spring, MD 20993.

Abstract
Human Immunodeficiency Virus (HIV) is a retrovirus
that causes AIDS (Acquired Immuno deficiency
Syndrome). The global pandemic of HIV-1 affects all
ages, sexes, and races. As of December 2015,
approximately 36.7 million people worldwide are
living with HIV. HIV-1 is diversified as subtypes and
recombinant forms. Broadly, HIV-1 is divided into
groups: M (major), O (outlier), N (neither M nor O),
and P (a newly discovered virus subtype). HIV-1
Group M is responsible for the global HIV epidemic.
Within HIV-1 group M, there are distinct subtypes (A
to K) and recombinant forms. Previously it has been
reported that West central Africa is an epicenter for
circulation of diverse HIV-1 subtypes. Viral diversity
poses challenges with pathogenicity, diagnosis and
treatment. Therefore, there is need for continuous
surveillance for emerging viruses. Our goal was to (1)
sequence the near full length of HIV-1 virus and (2)
discover heterogeneity of HIV-1 in the infected
individuals, whether the subtypes evolved
independently or as evolved as recombinants of
subtypes. Our laboratory is evaluating samples from
Cameroon as a model to study HIV-1 diversity. The
rationale for selecting Cameroon is that these blood
specimens may contain complex HIV strains and other
retroviruses. Cameroon in West Central Africa is
known to harbor multiple genetically diverse and
recombinant strains but CRF02_AG continues to be
the pre-dominant strain for over a decade. However,
emergence of recombinants of CRF02_AG may be
likely as the epidemic evolves in this region which
may affect diagnostics, vaccine and therapies. We
characterize near full length HIV strains received from
infected plasma samples from Cameroon. In order to
Background
Human Immunodeficiency Virus (commonly referred to as HIV) is a global pandemic that affects over 6,850 people every day worldwide1. HIV-1 is
a retrovirus that causes Acquired Immuno Deficiency Syndrome (AIDS). The HIV virus can be divided into type 1 (HIV-1), which is the most
widespread type worldwide and is composed of further classifications, including the four groups: M (the major group; further classification of
Group M includes: subtypes A - K), N (neither M nor O), O (the outlier group), and P (which is a newly discovered virus subtype), and type 2 (HIV-
2). The virus is composed of three structural proteins, including Group-specific antigen (gag), Polymerase (pol), Envelope (env), which form the
genomic sequence of HIV. The virus integrates itself into the genome of the infected host and includes error-prone replication, which is the
underlying principle of why there is no cure for HIV. Blood specimens from Cameroon were collected in order to study the viral genetic diversity of
the HIV-1 virus and how the HIV-1 Virus subtypes evolve in the infected host. Circulating recombinant forms (CRFs) are highly diversified strains
that represent recombinant HIV-1 genomes. The evolution of HIV-1 recombinants poses a threat to diagnostics, vaccines, and treatment. Currently, Figure 1. HIV-1 Viral Structure
When HIV enters a cell, it completes reverse
the CRF, CRF02_AG, which is composed of subtypes A and G, is the most dominant strain in Cameroon. Recombinants of CRF02_AG may form as the transcription of its RNA genome. HIV uses the enzyme,
virus evolves. The primary purpose of this investigation is to sequence the genome of the HIV-1 virus and to discover heterogeneity of HIV-1 in the
called reverse transcriptase, to convert RNA to DNA. HIV
has two single strands of RNA for its genome. The core of
the HIV virus houses the viral genome, as well as enzymes
infected individuals, whether the subtype evolved independently or evolved as recombinants of subtypes. that the virus needs to replicate in the host cell.
Conclusions
Methods
Through this investigation, we were able to discover
the heterogeneity of HIV-1 in the infected individuals.
Library Preparation The genotypes CRF02 and F2 both co-evolved within
Sequencing Data Analysis Final Results
Add sequencing adapters the two partners [6541 (6) and 6542 (5)].
Transfer libraries to the Process/Annotate data, Interpret and report
and prepare libraries for biological context The two female partners [6542 (5) and 6541 (6)]
flow cell and sequence report genomic variants
sequencing were infected with the F2 recombinant with CRF02
(F2/CRF02). CRF02 part of the genome may be
contributed by their partner.
Flow Chart of Computational The Results and Analysis are shown
Nextera XT 1 Tagmentation Methodology The total reads, shown in Table 1, displays the
DNA Library Prep Workflow below, in the Results Section.
number of times a nucleotide is read during the
2 Barcoding PCR
Import Raw sequences to CLC

accomplish this goal, the nucleic acids were extracted Genomic Workbench v8.5
Table 1. Reads Summary
from the plasma. Then, the reverse transcription of 1 sequencing process. The number of reads evidently
the RNA was performed using two gene specific pri- Check the quality of reads/cross
contamination
Reads Summary supports the dominant genotype in each patient.
mers to obtain full length HIV-1 cDNA. Polymerase Percent It is interesting to note that in the male partner: the
Chain Reaction (PCR) was performed to amplify the 2 Generate simple countigs or map
Sample
Gender PCR ID
Total Mapped
Mapping to Genotype

regions of the viral genome in order to contain a near reads to the reference sequence
ID Reads Reads
HXB2 (HIV-1) recombinant CRF02/F2 did not dominate, but
full-length genome of HIV-1. Amplicon size was 6542(5) Female S1 2,979,459 965,351 32.40 F2/CRF02 rather CRF02 is the predominant genotype.
confirmed by running 1% Agarose gel electrophoresis. Generate and validate consensus
sequence of the alignment 6541(6) Female S2 3,560,080 1,700,492 47.77 F2/CRF02
In this study, we did not observe subtype F2 and
Sequencing libraries are generated from PCR
CRF02 evolve independently in the female partners.
6544(5) Male S3 3,505,956 2,670,768 76.18 CRF02
amplicons following Nextera XT (Illumina) Align/Blast to published references
Table 1 displays the three analyzed HIV-1 Finding the genotype of infected HIV patients can
manufacturer protocol. MiSeq Next-Generation
infected patient samples, including their
Sequencing (NGS) technology was used in order to help acquire and target new drugs and vaccines
complete whole-viral genome sequencing. Our
Determine genotype or Variants
(SNP) call and annotate sample IDs, History and Genotype.
preliminary results indicate that out of the 3 HIV engineered to a specific genotype, such as F2 or the
infected linked samples evaluated, one male sample recombinant form F2/CRF02.
was infected with CRF02_AG and two of his partners
were infected with F2 recombinant with CRF02. Figure 2. HXB2(HIV-1) mapping and Genome coverage >10,000x Analysis
CRF02 virus from the male partner recombined with Results In Figure 3, each of the patient samples was blasted against
F2 infected female partners and evolved as F2/CRF02 HIV-1 reference sequences (Subtypes A - K, CRF01, and
Figure. 2 Graphical CRF02). The highest scored reference was indicated in the
recombinants. The poster provides the specific References
methodological aspects of this work. This study representation of patient sample respective graphs. The x-axis represents the position in the
provides a useful tool in order to thoroughly (S1 to S3) reads mapped to genomic sequence, while the y-axis shows the frequency of the 1: H. Khalsa, Henry Francis, and Rafael Mazin.
understand the HIV epidemiology and pathogenesis, HXB2 (HIV-1 reference strain). base. Fig. 3A displays Sample 1, with recombinant CRF02/F2 as Bloodborne and Sexually Transmitted Infections in
HIV diagnosis, therapy, and future vaccine strategies. Coverage peak for each sample the predominant subtype. Fig. 3B displays Sample 2, also with
Drug Abusers in the United States, Latin America, the
was indicated in pink color the recombinant CRF02/F2 as the dominant subtype, while Fig.
across full length of the genome. 3C displays Sample 3, with subtype F2 as the lead subtype.
Caribbean, and SpainClin Infect
Dis. (2003) 37 (Supplement 5): S331-
Fig.3A,S1 Fig.3B,S2 Fig.3C,S3
S337 doi:10.1086/377543

Acknowledgements 2: Davey M. Smith, Douglas D. Richman, Susan J. Little

Dr. Viswanath Ragupathy., PhD (Mentor) & J Infect Dis. 2005 August 1; 192(3): 438444. Published
Dr. Indira Hewlett (Lab Chief)., PhD online 2005 June 30. doi: 10.1086/431682.
Food and Drug Administration (FDA)
3: Ragupathy V, Zhao J, Wood O, et al. Identification of
Lab of Molecular Virology (Building 72, Room 4230) new, emerging HIV-1 unique recombinant forms and
Office of Blood Research & Review drug resistant viruses circulating in Cameroon.
Center for Biologics Evaluation and Research Virology Journal. 2011; 8:185. doi:10.1186/1743-422X-8-
10903, New Hampshire Ave, Silver Spring MD 20993 185.
E-mail: viswanath.ragupathy@fda.hhs.gov