Running head: NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 1

NextGeneration Sequencing to Characterize Near Full Length Human

Immuno Deficiency Virus (HIV-1) Subtypes Evolving Within Infected Host

Supraja Kanipakam

Dr. Viswanath Ragupathy

Food and Drug Administration (FDA)

NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 2
Abstract

Background. 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. 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.

Methodology. Our laboratory is evaluating samples from Cameroon as a model to study HIV-1
diversity. Using infected plasma samples from Cameroon, we characterize near full length HIV
strains received from Cameroon. In order to accomplish this goal, the nucleic acids were
extracted, the reverse transcription of the RNA was performed using two gene specific primers to
obtain full length HIV-1 cDNA, and polymerase chain reaction (PCR) was performed to amplify
the products. Amplicon size was confirmed by running 1% Agarose gel electrophoresis.
Sequencing libraries are generated from PCR amplicons following Nextera XT (Illumina)
manufacturer protocol, along with MiSeq Next-Generation Sequencing (NGS) technology.

Results. Our preliminary results indicate that out of the 3 HIV infected linked samples evaluated,
one male sample was infected with CRF02_AG and two of his partners were infected with F2
recombinant with CRF02. CRF02 virus from the male partner recombined with F2 infected
female partners and evolved as F2/CRF02 recombinants.

Conclusion. This study provides a useful tool in order to thoroughly understand the HIV
epidemiology and pathogenesis, HIV diagnosis, therapy, and future vaccine strategies.

Keywords: HIV-1 subtypes, NextGeneration Sequencing, Characterize near full-length genome

NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 3

Introduction

Human Immunodeficiency Virus (commonly referred to as HIV) is a global pandemic

that affects over 6,850 people every day worldwide . 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), N (neither M nor O), O (the outlier group), and P (which is a
newly discovered virus subtype), and type 2 (HIV- 2). The M group comprises of sub-subtypes
(A-K) and circulating recombinant forms. Within group M, subtype B is the most disseminated
variant and is assumed to be the causative agent in 11% of all HIV cases worldwide (Junqueira
and Almeida, 2016). 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. Previously it has
been reported that West central Africa is an epicenter for circulation of diverse HIV-1 subtypes
(Magiorkinis et al., 2016). 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, 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 virus
evolves. According to Wu, viral diversity poses challenges with pathogenicity, diagnosis and
treatment. Therefore, there is need for continuous surveillance for emerging viruses. 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 infected individuals, whether the subtype evolved independently or
evolved as recombinants of subtypes.

Materials and Methods

Samples/Study population
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
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 4
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.

Procedure
In order to accomplish this goal, the nucleic acids were extracted from the plasma using the
Qiagen RNA Purification kit. Then, the reverse transcription of the RNA was performed using
two gene specific primers to obtain full length HIV-1 cDNA. The SuperScript III First-Strand
Synthesis kit was used to synthesize first-strand cDNA from total RNA primed with a gene-
specific primer. Polymerase Chain Reaction (PCR) was performed to amplify the regions of the
viral genome in order to contain a near full-length genome of HIV-1. In order to confirm the
Amplicon size, gel electrophoresis was conducted with 1% Agarose gel. Sequencing libraries are
generated from PCR amplicons following Nextera XT (Illumina) manufacturer protocol. Next,
libraries were then transferred to the flow cell for sequencing. MiSeq Next-Generation
Sequencing (NGS) technology was used in order to complete whole-viral genome sequencing
and provide a comprehensive view of the entire genome. Ultimately, data analysis was
completed to process/annotate the data, report genomic variants and biological context.
.

Results

Figure 2. HXB2(HIV-1) mapping and Genome coverage >10,000x

Figure. 2 Graphical representation

In Figure 3, each of of
thepatient
patientsample (S1was
samples to S3) reads
blasted mapped
against to HXB2
HIV-1 (HIV-1
reference sequences
reference strain). Coverage peak for each sample was indicated in pink color across full length of
(Subtypes A - K, CRF01, and CRF02). The highest scored reference was indicated in the
the genome.
Figure 3A. Sample 1 Figure 3B. Sample 2 Figure 3C. Sample 3
respective graphs. Fig. 3A displays Sample 1, with recombinant CRF02/F2 as the predominant
subtype. Fig. 3B displays Sample 2, also with the recombinant CRF02/F2 as the dominant
subtype, while Fig. 3C displays Sample 3, with subtype F2 as the lead subtype. Samples 1 and 2
are re-combinants, while Sample 3 is a pure genomic form of the F2.
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 5

Conclusion
Through this investigation, we were able to discover the heterogeneity of HIV-1 in the infected
individuals. The genotypes CRF02 and F2 both co-evolved within the two partners [6541 (6) and
6542 (5)]. Additionally, the reads were calculated for each patients samples.

Table 1. Reads Summary

Reads Summary

Sample Gender PCR ID Total Mapped Percent Genotype

ID Reads Reads Mapping to
HXB2 (HIV-1)

6542(5) Female S1 2,979,459 965,351 32.40 F2/CRF02

6541(6) Female S2 3,560,080 1,700,492 47.77 F2/CRF02

6544(5) Male S3 3,505,956 2,670,768 76.18 CRF02

Based on this studys results, the following conclusions can be made:

The two female partners [6542 (5) and 6541 (6)] were infected with the F2 recombinant
with CRF02 (F2/CRF02). CRF02 part of the genome may be contributed by their partner.

The total reads, shown in Table 1, displays the number of times a nucleotide is read
during the sequencing process. The number of reads evidently supports the dominant
genotype in each patient.

It is interesting to note that in the male partner: the recombinant CRF02/F2 did not
dominate, but rather CRF02 is the predominant genotype.

In this study, we did not observe subtype F2 and CRF02 evolve independently in the
female partners.

Finding the genotype of infected HIV patients can help acquire and target new drugs and
vaccines engineered to a specific genotype, such as F2 or the recombinant form
F2/CRF02.
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 6

Discussion
In this present study, the various plasmas were samples taken from patients in West Cameroon.
The RNA, which was extracted from the plasmas using the nucleic acid extraction protocol, was
used to develop the assay. According to Nadiye, plasma representing a range of viral loads was
used to develop the assay. Then, RNA was extracted from the plasma and reverse-transcribed,
using two gene-specific primers, which was performed to the present study. In another study, a
PCR assay for the detection of HIV-1 group M proviral DNA in whole blood was developed and
used a double-stranded primer (DSP), which allows rapid amplification. With the PCR
amplification, both the forward and reverse primers are extended to form the double-stranded
product, which is the final result the study aimed to obtain (Pau, 2010). Ultimately, PCR was
conducted for amplification across the HIV genome and sequencing occurred using an automated
sequencer, which was conducted in both studies, along with the present study.

Acknowledgements
The author of this scientific paper wishes to acknowledge her mentor, Dr. Viswanath Ragupathy,
and the lab chief Dr. Indira Hewlett at the Food and Drug Administration (commonly referred to
as FDA), specifically in the Lab of Molecular Virology within the Center for Biologics
Evaluation and Research. These findings and conclusions in this paper have not been formally
released by the Food and Drug Administration and should not be construed to represent any
Agency determination or policy.

Works Cited

Junqueira, D.M., & Almeida, S. M. (2016). HIV-1 subtype B: Traces of a pandemic. Virology,
495173-184. doi: 10.1016/j.virol. 2016.05.003

Magiorkinis, G., Angelis, K., Mamais, I., Katzourakis, A., Hatzakis, A., Albert, J., & ...
Paraskevis, D. (2016). Research paper: The global spread of HIV-1 subtype B
epidemic. Infection, Genetics And Evolution, doi:10.1016/j.meegid.2016.05.041

Nadai Y, Eyzaguirre LM, Constantine NT, Sill AM, Cleghorn F, Blattner WA, et al. (2008).
Protocol for Nearly Full-Length Sequencing of HIV-1 RNA from Plasma. PLoS ONE
3(1): e1420. doi:10.1371/journal.pone.0001420
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 7

Pau, C., Wells, S. K., Rudolph, D. L., Owen, S. M., & Granade, T. C. (2010). A rapid real-time PCR assay
for the detection of HIV-1 proviral DNA using double-stranded primer. Journal Of Virological
Methods, 164(1-2), 55-62. doi:10.1016/j.jviromet.2009.11.027

Wu JW, Patterson-Lomba O, Novitsky V, Pagano M. (2012). A Generalized Entropy Measure of Within-

Host Viral Diversity for Identifying Recent HIV-1 Infections. Rosenthal. K, ed. Medicine.
2015;94(42):e1865. doi:10.1097/MD.0000000000001865

Annotated Bibliography

Averting HIV and AIDS. (n.d.). Retrieved from http://www.avert.org

This website from the global organization, AVERTing HIV Strains and Types, gives It
includes various sections/divisions, including information about the Human Immunodeficiency
Virus (HIV) virus, transmission and prevention, and testing of HIV. The webpage about the HIV
virus primarily includes background information regarding the various strains and types of the
HIV virus. Under the broad HIV virus are two types: HIV-1 virus and the HIV-2 virus. The
webpage states that worldwide, the predominant virus is HIV-1, while HIV-2 virus, which is
fairly uncommon, is concentrated mostly in West Africa. Also, HIV-2 virus progresses slower
than HIV-1 virus. The webpage mentions that the strains of HIV-1 can be classified into four
groups: M (the major group), N, O, and P. The M group is responsible for the majority of the
global HIV epidemic. The other three groups are quite uncommon and only occur in Cameroon,
Gabon and Equatorial Guinea. The transmission emphasizes that HIV can easily be spread to
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 8
others through contaminated blood transfusions and organ transplants, unprotected sexual
intercourse, injecting shared equipment, including needles and syringes, used to prepare and
inject drugs with someone who has HIV, and can be passed from mother-to-baby during
pregnancy, childbirth and breastfeeding. For prevention, the website mentions that everyone is at
risk at getting HIV, but some people have a higher risk of getting HIV because they engage in
certain activities (e.g. injecting drugs) that are more likely to transmit the virus. Under the
Testing of HIV section, the website mentions that the HIV virus can only be detected after a
window period, which is dependent on the specific type of HIV test one takes.
This webpage is very helpful to my research, because it not only allows me to have
background information with regards to the classification of the HIV-1 virus, but also allows me
to acquire a more in-depth understanding of the other 3 subgroups of HIV (Groups N, O, and P).
Additionally, through this website, I learned that certain subtypes have a greater risk of
transmission than others and that antiretroviral drugs (ARVs) are generally effective against a
wide range of subtypes of HIV. It was interesting to see how this website states that the window
period refers to the time it takes for HIV to show up in a test, for HIV, as I thought that it was 15
days regardless of which HIV test an infected patient takes. Through the information on this
website, I was able to gain more insight and knowledge into the specifics of the HIV virus,
including its classifications, transmission, prevention, and testing.

Divino, F., de Lima Guerra Corado, A., Gomes Naveca, F., Stefani, M. A., & Bello, G. (2016).
High Prevalence and Onward Transmission of Non-Pandemic HIV-1 Subtype B Clades in
Northern and Northeastern Brazilian Regions. Plos ONE, 11(9), 1-14.
doi:10.1371/journal.pone.0162112

This article discusses the background of the Human Immunodeficiency Virus type-1
(HIV-1) and how subtype B is prevalent. The study primarily aimed to estimate the prevalence of
the HIV-1 Subtype B pandemic across different Brazilian regions. According to the study, about
780,000 people were living with the Human Immunodeficiency Virus Type 1 (HIV-1) in Brazil in
2014. The incidence is divided into the regions in Brazil, as most Brazilian cases were
concentrated in the Southeastern region (48%), followed by the Southern (22%), Northeastern
(17%), Northern (7%) and Central- Western (6%) regions. The study used this data to focus
mainly on the HIV-1 subtype B. The researchers in the study obtained HIV-1 subtype B pol
sequences from Brazil and used the samples for phylogenetic analysis, where HIV-1 Brazilian
sequences were aligned with subtype B pol (PR/RT) sequences from the United States and were
thus compared. The results yielded that highly variable prevalence across locations, as there is a
large proportion (41%) of HIV-1 subtype B infections in the state of Roraima. When analyzed by
Brazilian region, the highest proportion of BCAR sequences was observed in the Northern
(17%), followed by the Northeastern (4%), Central-Western (1%), Southeastern (1%) and
Southern (1%) regions.
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 9
This study is helpful to my research because of its statistics and facts regarding the
prevalence of subtype B in Northern and Northeastern Brazilian Regions. The study provides a
table with the region in Brazil and state along with the specific sampling time. This study has
helped me understand the relationship between sequences and the incidence and/or prevalence of
the HIV infection. I also gained insight about Brazilian HIV incidence and pandemic across the
various regions in Brazil, including the Southeastern region, Southern, Northeastern, Northern,
and Central- Western regions.

FDA Vaccines, Blood & Biologics. (n.d.). Retrieved from http://www.fda.gov/BiologicsBlood

Vaccines/default.htm

The Food and Drug Administration (FDA) Vaccines, Blood and Biologics
department/division primarily includes information regarding blood components, gene-based
treatments and cell-based treatments. This information is more regulatory, than scientific, as it
includes information regarding blood establishments and guidance and biologic forms. The
Center for Biologics Evaluation and Research (CBER) primarily works with all biologics
research, including blood components, by regulating the collection of blood and developing and
enforcing quality substances and cellular therapy products and human gene therapy products, by
regulating cell and gene therapy. Specifically, in the division of cellular and gene therapy
products, the website explains the differences, as cellular therapy products include cellular
immunotherapies, and autologous and allogeneic cells for certain therapeutic indications, while
human gene therapy typically treats a disease or abnormal medical condition, by introducing
genetic material into a persons DNA to replace faulty or missing genetic material.
Through the FDA website, I was able to explore different categories under the branch of
Vaccines, Blood and Biologics, that I did not previous know about. Additionally, I was able to
learn additional information about the significance of the center that my internship takes place in,
which is known as the Center for Biologics Evaluation and Research (CBER). A list consisting of
all Donor Screening Assays for HIV Diagnostic Assays is provided on the website and can be
referred to use specific effective assays that would yield accurate results. Additionally, I learned
about cellular therapy and human gene therapy. I can now understand when scientists and
researchers use such terminology in the CBER center/division of FDA. Therefore, this resource
is of help and use for my research. However, this website does not provide specifics about HIV
regulations or treatment regarding the HIV virus (the use of antiretroviral drugs).

Hewlett, Dr. Indira. Personal interview. 23 Oct. 2016.

Dr. Indira Hewlett is the principal investigator (PI) and chief of the Office of Blood
Research and Review (OBRR), Division of Emerging and Transfusion Transmitted Diseases
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 10
(DETTD) at the Laboratory of Molecular Virology (LMV). Dr. Hewlett graduated from
University at Albany in New York in 1980. About 3 years ago, Dr. Hewlett was affiliated with
the National Institutes of Health (NIH), until the lab was moved to its current location at FDA.
Dr. Hewlett works for the Deputy Director, Dr. Paul A. Mied, who is the subordinate for Dr. Hira
L. Nakhasi. In addition to being the laboratory chief, she is also a microbiologist at FDA. In Dr.
Hewletts lab, the primary focus is placed on Human Immunodeficiency Virus (HIV) diagnosis
and pathogenesis and genetic diversity of the HIV virus.

HIV & AIDS Information : HIV Basics - HIV & AIDS. (n.d.). Retrieved from
http://www.aidsmap.com/hiv-basics

The NAM AidsMap webpage provides information regarding the Human

Immunodeficiency Virus (HIV), specifically about viral factors affecting HIV transmission and
the various subtypes of HIV. It mentions that there are three major HIV-1 groups: M (main), O
(outlier), and N (non-M/non-O). Statistically, the website states that about 90% of HIV-1
infections are classified as group M and these are distributed worldwide. Within the HIV-M
group, there is a further division into at least ten sub-subtypes. The webpage provides a list of the
distribution/proportion of HIV-1 sub-subtypes of the M group, according to the geographic
patterns. Additionally, 'circulating recombinant form' (CRFs), which are hybrid viruses that are
formed by different subtypes combining genetic material, are explored. For example: the
recombinant forms can occur between two subtypes, such as subtype A and subtype B, that co-
link or recombine together. Additionally, the website provides many resources to learn about the
distinct difference between HIV and AIDS, the symptoms of HIV, and the preexistence of HIV. It
states that AIDS stands for acquired immune deficiency syndrome, which develops when
someones immune system has been damaged by HIV. So, evidently HIV causes AIDS, as the
AIDS syndrome is passed on from the HIV infection. A common symptom among many people
is a short, flu-like illness, also known as a seroconversion illness, soon after an individual is
infected with HIV. On the contrary, the website states that it is possible for an HIV-infected
patient to not to have any symptoms at all, while the virus causes damage to the immune system.
Additionally, the website emphasizes that many individuals may feel typical symptoms,
including fever, cold, sore throat, etc., but one should take the HIV-1 test in order to know
whether he or she is positive or negative for the HIV-1 virus.
Through the information provided in the website, I learned that subtype B is the most
common subtype in many places, including West and Central Europe, the Americas, Australia,
South America, and several southeast Asian countries (Thailand, and Japan), as well as northern
Africa and the Middle East. This website allowed me to gain background information regarding
the subtypes and recombinants and its respective geographic areas. Additionally, I have a better
understanding of HIV-1 classifications, into subtypes and sub-subtypes within the four main
groups. I also understand the relationship/association between HIV and AIDS, as HIV causes
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 11
AIDS. This is useful to my research, because, through this source, I have acquired and
understood the basics of the HIV virus.

Hood, J. (2011). HIV/AIDS, health and the media in China (1st ed.). New York, NY: Routledge.
This book discusses the prevalence and incidence rates of HIV/AIDS in China. The
author of this book, Johanna Hood, is a member of the China Research Centre at the University
of Technology, Sydney, Australia. Over 500,000 individuals are affected every year by HIV in
China. Hood emphasizes the need for education in China, in order to inform individuals in China
prevention techniques and treatment. She mentions the need for antiretroviral drugs (ART),
which can effectively treat the HIV infection, by slowing the rate HIV makes multiplies in the
body, instead of using one medicine (monotherapy) to treat the HIV infection. Additionally, the
basics of transmission of HIV is included. The major modes the HIV virus can be transmitted
through contact with infected blood, sharing of needles/syringes, and sexual transmission. In
China, sexual transmission is the primary mode of transmission.
This book provides an interesting aspect related to HIV/AIDS, which is
prevention. Unlike the ways Averting HIV and AIDS suggested preventing HIV, Hood states
that an effective way to prevent individuals from acquiring the HIV infection is to deter the ways
in which the disease is portrayed in China and, instead, educate individuals using the correct
demeanor. The HIV basics of transmission are mentioned; this helped me gain insight as to how
HIV spreads in a HIV-1 infected patient (copies itself), along with its transmission route, and
prevention from the virus. The book includes pictures with the effect HIV has on the physical
body. This book helped me learn more about the basics of IV transmission and the physical
aspect of the HIV infection.

Junqueira, D.M., & Almeida, S. M. (2016). HIV-1 subtype B: Traces of a pandemic. Virology,
495173-184. doi: 10.1016/j.virol. 2016.05.003

This study centers in on subtype B of the Human Immunodeficiency Virus type 1 (HIV-1)
classification. The study states that subtype B is the most disseminated variant and is assumed to
be the causative agent in 11% of all HIV cases worldwide. The study discusses the HIV epidemic
and the diversification, by mentioning the four distinct groups of HIV, which include M (for
main), N (for non-M/non-O), O (for outlier), and P. According to the study, group M viruses
spread in a pandemic manner and are responsible for the majority of HIV infections worldwide,
while group N limits the effective spread of these viruses into humans. In addition to the main
groups, there is further classification with the sub-subtypes. The prevalent Group M includes
nine sub-subtypes (A, B, C, D, F, G, H, J, and K). Within some subtypes, high genetic variation
leads to the classification into sub-subtypes. The study emphasizes the importance of subtype B,
as most current HIV antiretroviral drugs were designed using HIV-1 subtype B. The article states
that, due to the evolution/prevalence of the HIV-1 subtype B, genetic variation within subtype B
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 12
has been found, as the pandemic strains of HIV type 1, group M, subtype B is varied depending
on the location; there is a Brazilian subtype B, which is different from the Korean and Thailand
subtype B. Subtype B is well-known for being the most widespread HIV-1 variant and for
accounting for approximately 11% of all infections worldwide.
This study provides useful background information regarding the specifics of HIV-1
subtype B, which is most prevalent subtype in all HIV-1 cases. Before reading this study, I had
basic preliminary knowledge that there were several sub-subtypes, but I did not know how these
sub-subtypes were formed or what led to their formation. I have learned, through this study, that
the high genetic variation led to the classification into sub-subtypes. The article includes a figure
regarding the estimated spatial dynamics of HIV-1 subtype B around the world, which helped me
understand the pathway and emergence of subtype B. I also learned about the subtype B genetic
variations, which vary according to the geographic location, which I did not know about before.

Magiorkinis, G., Angelis, K., Mamais, I., Katzourakis, A., Hatzakis, A., Albert, J., & ...
Paraskevis, D. (2016). Research paper: The global spread of HIV-1 subtype B
epidemic. Infection, Genetics And Evolution, doi:10.1016/j.meegid.2016.05.041

This article emphasizes the spread of the Human Immunodeficiency Virus type 1 (HIV-1)
group M subtype B globally. The virus was initially discovered in the 1980s and most of the
infections were classified as subtype B infections and were part of an epidemic that traveled
from Africa through Haiti to United States. This study utilizes a large dataset of global HIV-1
subtype B strains to map the spread of the subtype and describe the spread patterns. In order to
view these patterns, the researchers grouped the viral strains in geographic areas (including
North America, Europe, Central & South America, Caribbean, Africa, Asia and Oceania) using
phylogeographic analysis. This process allowed the researchers to discover which region had the
highest prevalence of HIV-1 subtype B strain. Additionally, an equation known as the Force of
Migration was used to summarize the export and import migration for the regions. Only the
European viral strains were used to infer migration routes among the European countries. It was
found that in Europe, the highest prevalence occurred in Western Europe, as over 70% of the
sequences were from this region alone. Thus, by using statistical phylogeography, the researchers
were able to identify migration patterns of subtype B around the globe. The results yielded that
HIV-1 spread through migration routes and the epidemic was caused primarily by geopolitical
factors, such as migration and tourism. In addition, the study found that their findings support the
argument that epidemic control policies incorporate political and socioeconomic factors.
This is helpful for my research, because subtype B is the subtype I will be investigating
to see if it is located in the patient sample. By learning the history and basics of subtype B, I will
be able to use the information for the research and further my knowledge regarding the subtype.
In addition, I learned about the effect the HIV-1 (subtype B) had on the Eastern Hemisphere,
including Europe, Unite Kingdom, France and Switzerland. The study states that subtype B is the
most prevalent in the Western World. This article has helped me understand the spread of the
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 13
HIV-1 subtype B infection around the world and its transmission globally. Also, I learned a new
equation that can be used to summarize the spread of data; the formula is known as Force of
Migration. It is interesting to note that one can view the spread of the HIV-1 infection across
different regions worldwide by simply using phylogeographic analysis, which indeterminably
determines evolution. I learned that phylogeographic analysis was developed by the process of
Polymerase Chain Reaction (PCR), which amplifies/replicates DNA segments. Phylogeographic
analysis reveals spatial genetic structure and is associated with evolution. Therefore, learning
more about subtype B and its history will benefit my research, as it will result in more
comprehension/understanding.

Nadai Y, Eyzaguirre LM, Constantine NT, Sill AM, Cleghorn F, Blattner WA, et al. (2008).
Protocol for Nearly Full-Length Sequencing of HIV-1 RNA from Plasma. PLoS ONE
3(1): e1420. doi:10.1371/journal.pone.0001420

This article explains the correct procedure and protocol for sequencing a near full-length
HIV-1 genome from a HIV-infected patients plasma/serum. The article provides a useful tool, in
order to discover which strategy utilizing different primers and different amplicon strategies
(two-amplicon strategy and three-amplicon strategy). To test the effectiveness and success of the
procedure, the viral loads, which are the number of copies of HIV in the blood, was measured.
Overall, a well-developed procedure was developed, which is condensed into 3 steps as follows:
(1) Plasma representing a range of viral loads is used to develop the assay. (2) RNA was
extracted from the plasma and reverse-transcribed, using two gene-specific primers (UNINEF 7
or VIF-VPUoutR1). (3) PCR was conducted for amplification across the HIV genome and
sequencing occurred using an automated sequencer (Applied Biosystems). It was found, based
on reliability and efficacy, that the QIAamp viral RNA mini assay was prime after comparisons
with other commercially available extraction assays. Using this kit allowed for an increase in
improved sensitivity for low VL samples. Data Table 2 displays the results that, for a viral load
of over 750,000 HIV-1 RNA copies/mL, the PCR regions, Env-Nef, Pol-Vpu, and Gag-Pol, had
an efficiency rate/percentage of 100 percent, which is a successful rate, according to the article.
Therefore, by following this protocol, one can successfully and effectively study the molecular
epidemiology of HIV-1 in regions of the world where only serum or plasma is available.
This procedure is an exemplary model for effective sequencing of the HIV-1 genome.
One concern that may arise is due to the fact that this article was published eight years ago; since
technology has improved and has increased in effectiveness, the machines mentioned in the
article are slightly outdated, as they have been replaced by more efficient utilities. However, the
amount (number of microliters) of each component used in this procedure can be applied to
accurately obtain the desired result, which is the near full-length HIV-1 genome. After viewing
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 14
the result and analysis of this study, I was able evidently see the efficiency rate and the success of
the mini assay kit. This is useful to my research because, when I am conducting the PCR process,
I can use the QIAamp viral RNA mini assay, as it both effective and reliable for accurate results.
Additionally, this article exposed to different terminology used in this particular field of research,
including viral load, which is the number/amount of HIV-1 RNA copies/mL in a HIV-infected
patients sample.

Ndiaye, O., Diop-Ndiaye, H., Ouedraogo, A. S., Fall-Malick, F. Z., Sow-Sall, A., Thiam, M.,
& ... Toure-Kane, C. (2015). Comparison of four commercial viral load techniques in an
area of non-B HIV-1 subtypes circulation. Journal Of Virological Methods, 222122-131.
doi:10.1016/j.jviromet.2015.06.002

This study primarily focuses on comparing four commercial viral load techniques with
HIV-1 subtypes, excluding subtype B. The four commercial viral load techniques were as
follows: Nuclisens EasyQ v2.0, HIV-1 V2.0 COBAS AmpliPreP/COBAS Taqman, Generic HIV
Charge Virale assay and Abbott Real Time HIV-1. In order to effectively test these kits, several
steps were taken. First, blood samples were collected from patients or blood donors and cells
separation was processed to obtain plasma within 4 h after collection. First, blood samples were
collected from patients or blood donors and cells separation was processed to obtain plasma
within 4 h after collection. Then, the viral quantitation was conducted for each technique. For the
NucliSENS EasyQ v2.0 (EQ) assay, nucleic acids were extracted from 200 uL of plasma and
Polymerase Chain Reaction (PCR) was completed to amplify the samples and obtain the
copies/mL from the plasma, which yielded to be range between 1.37.0 log10 copies/ml. For the
HIV-1 V2.0 COBAS AmpliPreP/COBAS Taqman, Reverse-Transcription (RT) was conducted,
along with PCR to obtain an amplified region in the gag gene of the genome. Additionally,
nucleic acid was extracted and amplification allowed a range between 1.37.0 log10 copies/ml.
The Generic HIV Charge Virale assay used manual RNA extraction and amplification, yielding
a range between 2.40 7.0 log10 copies/mL from plasma. Lastly, the Abbott Real Time HIV-1
used amplification in the integrase region of the pol region and obtained a range between 1.60
7.0 log10 copies/mL for the plasma. Through these steps, the researchers were able to compare
VL quantitations and concluded that the highest performance, along with repeatability,
reproducibility, and accuracy, was yielded by Abbot Real Time HIV-1.
This study allowed to gain insight and knowledge about testing the viral load, as I
complete the PCR process with the samples I am given in the lab, but do not know how to obtain
the viral load. This study has helped me understand the procedure and how to compare and
contrast different viral load techniques. It can be deduced from the experiment that Abbott Real
Time performance provides the highest performance. I may be able to use the Abbott Real Time
performance to obtain a high viral load, that is precise and accurate.
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 15
Pau, C., Wells, S. K., Rudolph, D. L., Owen, S. M., & Granade, T. C. (2010). A rapid real-time
PCR assay for the detection of HIV-1 proviral DNA using double-stranded
primer. Journal Of Virological Methods, 164(1-2), 55-62.
doi:10.1016/j.jviromet.2009.11.027

This study uses a rapid real-time Polymerase Chain Reaction (PCR) assay to detect HIV-
1 proviral DNA in whole blood. The PCR reaction amplifies the DNA strands, to create a
significant amount of copies. Real-time PCR technology allow for faster amplification and is
more applicable to the confirmation of the HIV-1 infection. The co-amplification of a human
gene RNase P was the internal control, in this experiment, to monitor the efficiency of the DNA
extraction and PCR amplification. RNase-P is a form of ribonuclease that processes the 5-ends
of precursor-tRNAs and also acts like a catalyst. In addition, real-time PCR monitors the
amplification of a targeted DNA molecule during the PCR process. The HIV-1 amplification was
considered to be fully effective. In this study, a PCR assay for the detection of HIV-1 group M
proviral DNA in whole blood was developed and used a double-stranded primer (DSP), which
allows rapid amplification. With the PCR amplification, both the forward and reverse primers are
extended to form the double-stranded product, which is the final result the study aimed to obtain.
All forty-two HIV-1 whole blood specimens (including sero-positive and sero-negative
specimens) tested were classified correctly by this PCR assay. The study states that this PCR
assay may be useful as an alternative confirmation test in HIV testing.
This study provides information regarding what occurs during the amplification process
with the Polymerase Chain Reaction. The diagram shows the forward primer and reverse
primers interaction and the final product after the products have been amplified. This helps me
comprehend how the PCR amplification works internally. Additionally, I now understand the
lengths of the DNA and the corresponding lengths of the primers (forward and reverse), as the
forward primer starts from 5 to 3, which the reverse primer starts from 3 to 5. The reverse
primer is complementary to the forward primer. Therefore, during PCR, the forward primer and
reverse primer both extend to the form the double-stranded product. This PCR assay is effective
and efficient, in that it utilizes real-time PCR. Additionally, before the PCR process, I typically
complete a pre-PCR procedure, in which I add a primer. This study displayed the use of a primer
for rapid real-time PCR, which is similar to my research. This article has helped me review the
proper/correct procedure when doing experiments in the lab. An important concept I learned was
that by amplifying a short target sequence by removing the probe-binding sequence between the
primers, it will result in high amplification efficiency and short PCR cycles.

Ragupathy, Dr. Viswanath. Personal interview. 13 Oct. 2016.

Dr. Viswanath Ragupathy, my mentor, works as a scientist with primary focus on

infectious diseases, at the Food and Drug Administration (FDA) in Silver Spring, Maryland for
approximately 9 years. Within FDA, Dr. Ragupathy works in the Department of Health and
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 16
Human Services (HHS) in the Center for Biologics Evaluation and Research. Specifically, in
CBER, Dr. Ragupathy works in the Office of Blood Research and Review (OBRR) in the
Laboratory of Molecular Virology (LMV). In 1995, Dr. Ragupathy completed Master of Science
(MS) in Biochemistry at Annamalai University, which is located in Chidambaram Area, India.
Afterwards, in 2006, he completed his Ph.D in Bio Medical Sciences at Dr MGR Medical
University, which is located in the state of TamilNadu, India. Since 2007, Dr. Ragupathy has
been working as a scientist at FDA. Dr. Ragupathy has 28 publications, most of them relating to
the Human Immunodeficiency Virus type 1 (HIV-1) detection or identification of other emerging
infectious diseases.

Sakkhachornphop, S., Kijak, G. H., Beyrer, C., Razak, M. H., Sanders-Buell, E., Jittiwutikarn, J.,
& ... Tovanabutra, S. (2015). An effective tool for identifying HIV-1 subtypes B, C,
CRF01_AE, their recombinant forms, and dual infections in Southeast Asia by the multi-
region subtype specific PCR (MSSP) assay. Journal Of Virological Methods, 21770-78.
doi:10.1016/j.jviromet.2015.02.015

This article was published in the Journal Of Virological Methods and emphasizes the use
of a multi-region subtype specific (MSSP) PCR. It states that multi-region subtype specific
(MSSP) PCR is an effective and simple tool to identify subtypes of the HIV-1 virus. The MSSP
assay is described, by the article, as a PCR strategy that amplifies eight short regions and the
HIV-1 genome using subtype-specific primers. This study focuses primarily in Thailand, India
and China to test the MSSP assays ability. Most of the study is based off of Thailand, as a panel
of 41 clinical DNA samples were obtained primarily from northern Thailand was used to test the
assay performance. The results yielded that the performance of the assay on the full-length HIV-
1 genome provided 100% specificity overall. The overall sensitivity of the MSSP assay among
the following genes: Gag (p17-p24), Pol (p2-p7-p6 protease), Pol (p51 RT), Pol (p31 integrase),
Vpr, Env (gp120), Rev and Nef was 90%. Additionally, this study provided the distribution of
subtypes across different HIV-1 infected patient samples. Subtype distribution was among the
following subtypes/recombinant forms: CRF01_AE, subtype B, CRF01_AE/B recombinant, and
CRF01_AE/C recombinant. This study utilized a genotyping tool to capture the recombinant
genomes and dual infections. The genotyping tool allowed for graphs and charts displaying the
various subtypes and recombinant forms within a HIV-1 infected patient. This study can
primarily be used for vaccine development and evaluation.
This study provides various displays, in the form of graphs, diagram of MSSP
assay layout, visual of the genome structure, and an image of the gel profile, which clearly
showed the band size for the amplified product with the subtype-specific primer for recombinant
form CRF01 AE, and subtypes B and C. This helped me understand the effectiveness and
capability of the MSSP assay. I was able to further my knowledge regarding recombinant forms,
which are subtypes that co-evolve and fuse with their DNA. Essentially, I learned that HIV-1
infected patients with a recombinant forms, means that they have tow strains of HIV that
 NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 17
combined together. In order to find these subtypes and recombinant forms, PCR was completed
using the MSSP assay. It is interesting to note that the multi-region subtype specific (MSSP)
assay was 10% lower than the RNA mini assay provided by Qiagen. Perhaps, by using this assay,
I will be able to accurately amplify the products and gain the correct band size for the gene-
specific primers that I use.
U
U Wenhui Hu, Rafal Kaminski, Fan Yang, Yonggang Zhang, Laura Cosentino, Fang Li, Biao Luo,
David Alvarez-Carbonell, Yoelvis Garcia-Mesa, Jonathan Karn, Xianming Mo, and Kamel
Khalili, RNA-directed gene editing specifically eradicates latent and prevents new HIV-1
infection. PNAS 2014 111 (31) 11461-11466; doi:10.1073/pnas.1405186111.

This article discusses a possible way to eradicate the HIV -1 genome and be able to
prevent a new HIV-1 infection. First, the researchers eliminated the integrated HIV-1 genome by
using the Cas9/guide RNA (gRNA) system. Cas9 is a nuclease that catalyzes site-specific region
of double stranded DNA. In this study, Cas9 system has effectively disrupted the HIV-1 genome
in latently infected cells, suppressed viral gene expression and replication, and immunized
uninfected cells against HIV-1 infection. Then, the researchers used the Cas9 method to discover
whether it would be able to eradicate integrate HIV-1 DNA. The Cas9 method proved to be
capable with high efficiency. Additionally, the Cas9 cells may be able to be used in the future in
place of antiretroviral drugs, as they can be engineered to be specific, effective, and efficient.
This study includes a new theory regarding eliminating latent and preventing a new HIV-
1 infection. It contains graphs of the viral loads of different samples and a picture of the gel
electrophoresis profile, which indicates the band length of the DNA. It also displays the HIV
genome, describing the exact locations/positions in the genome. This helped me to visually
understand the steps the author of this study was taking. I was able to learn that the
regions/nucleotide positions correspond to the near full-length HIV genome.

Wu JW, Patterson-Lomba O, Novitsky V, Pagano M. (2012). A Generalized Entropy Measure of

Within-Host Viral Diversity for Identifying Recent HIV-1 Infections. Rosenthal. K, ed.
Medicine. 2015;94(42):e1865. doi:10.1097/MD.0000000000001865

This article discusses a study that was conducted in order to see whether the change in the
diversity of the HIV-1 virus over time varies across the different regions of the viral genome. An
entropy measure, which is a measured change is temperature, is recorded to discover the viral
diversity. The study utilizes a HIV incidence assay, which allows characterization of HIV
epidemics, and assessing incidence of HIV. The study acquires the structural gag gene of the
HIV-1 genome by using single genome amplification followed by direct sequencing. The study
focused on the gag gene primarily because gag is a viral protein that is more favorable with
disease prognosis and can induce T cell responses into the cells associated with control of viral
replication. This process resulted in a large sequence of 1518 nucleotides in length. Different
NEXTGEN SEQUENCING TO CHARACTERIZE HIV-1 SUBTYPES 18
nucleotide positions were chosen to calculate the entropy. The points were graphed according to
the respective nucleotide position and entropy measurement. This allowed the researchers to
view the development of within-host diversity over time at different nucleotide regions.
This study is helpful for my research, because of its information about the methods of
research and the protocol provided. The study states that in order to effectively analyze the viral
diversity of the HIV-1 virus, one must sequence a specific structural gene of the entire HIV-1
genome and compare it to another prominent gene in the genome. After acquiring the sequenced
genes, the study was able to obtain results and find the entropy measurements, which allowed the
study to measure the viral diversity. In addition, the study provides graphs regarding the entropy
across the genes in the genome, based on the position in the gene. This helps me to understand
the effect entropy has on the viral genome of the HIV-1 virus.