The document discusses dengue virus, which is transmitted by mosquitoes and causes dengue fever or dengue hemorrhagic fever in humans. It generated a phylogenetic tree of dengue virus serotypes 1, 2, and 3 isolated from different geographic regions to examine their relationships. The tree showed that serotypes 1 and 3 were restricted to Asia while serotype 2 was found in both Asia and Central America, supporting some geographic restriction of the serotypes. It concluded that analyzing all four serotypes could provide a more comprehensive understanding of dengue virus phylogeny and global spread.

1. Kellie Watkins Disease Modeling – Dengue Virus 2 May 2016
Dengue virus is a vector-borne arbovirus that is transmitted between humans primarily by
a species of mosquito known as Aedes aegypti. The first recognized epidemic of the disease
began in Southeast Asia during the 1950s and remained relatively confined to the region until the
global disruption caused by World War II, which is believed to have contributed to the
emergence and spread of the virus to new countries through the transport of infected humans
and/or mosquito vectors. Dengue is now endemic to over 100 countries, primarily tropical and
sub-tropical regions in aligning with the habitat of its vector, and it is estimated to cause about
390 million global infections per year among humans
(http://www.who.int/mediacentre/factsheets/fs117/en/).
Four serotypes of the single-stranded dengue RNA virus have been identified (DENV-1,
DENV-2, DENV-3, DENV-4), and the virus genome codes for three structural proteins known as
the capsid (C), envelope (E), and membrane (M) proteins. Despite their genetic relatedness,
infection by one dengue serotype does not confer protective immunity against the other serotypes
(http://www.cdc.gov/dengue/epidemiology/index.html). In humans, the virus can cause dengue
fever, or, in its more severe form, it can cause dengue hemorrhagic fever (DHF).
PROBLEM
A phylogenetic tree of DEN-1, 2, and 3 virus isolates was generated to examine the
geographic distribution of the isolates in each strain to assess whether: 1) infections of each
serotype were restricted to a geographical region or 2) multiple serotypes caused infections in the
same region.
It was hypothesized that if the serotypes were geographically restricted to certain regions,
then the phylogeny would reflect the following trend:

2. Kellie Watkins Disease Modeling – Dengue Virus 2 May 2016
1)
2)
The dengue virus’ genome is available through the National Center for Biotechnology
Information of the National Health Institutes. Through a sequence database known as GenBank,
under the Virus section, the nucleotide and protein sequences are available for extraction.
Queries for Type, Disease, Host, Region/Country, and Genome Region can be performed,
including collection date and release data parameters. For this project, multiple queries were
Geographic region 1
(Countries A, B C)
Geographic region 2
(Countries D, E, F)
Geographic region 2
(Countries G, H, I)
DENV-1
DENV-2
DENV-3
Geographic regions 1, 2, and/or 3
(Countries A, B, C, D, E, F, G, H, and/or I) DENV-1
DENV-2
DENV-3
Geographic regions 1, 2, and/or 3
(Countries A, B, C, D, E, F, G, H, and/or I)
Geographic regions 1, 2, and/or 3
(Countries A, B, C, D, E, F, G, H, and/or I)

3. Kellie Watkins Disease Modeling – Dengue Virus 2 May 2016
conducted to find one which produced a reasonable amount of sequences (i.e., ones that can be
analyzed without expending too much computational power). The final selection was, in the
order of Type, Disease, Host, Region/Country, and Genome Region, “1 2 and 3, known, any,
any, E,” without any specified collection dates or release data parameters specified.
Once the final query was selected, the FASTA file was downloaded and converted to a
MEGA file. It was aligned in Bioedit before performing the “Neighbor-joining” statistical
method. The test of phylogeny was the bootstrap method with the number of bootstrap
replications set at 1000. The substitutions type was nucleotide with the model/method as
Tajima-Nei. All substitutions were included with gaps/missing data treatment set as complete
deletion. Figure 2 summarizes these details:
Figure 2. Analysis Preferences

4. Kellie Watkins Disease Modeling – Dengue Virus 2 May 2016
The following tree was produced as Figure 3:
Figure 3.

5. Kellie Watkins Disease Modeling – Dengue Virus 2 May 2016

6. Kellie Watkins Disease Modeling – Dengue Virus 2 May 2016
From this tree, Group B (DENV-1) is sister to Group C (DENV-3). Group A (DENV-2)
is sister to Group B/C. Based on the region/country information, Group B and C are restricted to
Asia while Group A is a mixture of Asia and Central America. The host for all groups was
humans (Homo sapiens). Within Group B, there seemed to be a cluster of more recent collection
dates. These results, specifically focusing on bootstrap values greater than 70%, demonstrate
that Group B and Group C were circulating in Asia although Group A is also present in Central
America.
Some part of the genetic history of the virus supports these results. Studies indicate that
the DEN-1 and DEN-2 circulated in Central America and Africa around the 1970s while all four
serotypes circulated in Southeast Asia. Now, it is widely accepted that all four serotypes
circulate globally around the tropical or sub-tropical regions. Given this information, future
studies might find it valuable to analyze all serotypes for a more comprehensive look at the
phylogeny. Furthermore, a distinct range of collection dates might provide more insight to the
timescale of these genetic variations, providing a broader picture of its global presence in more
recent years. However, limitations exist with conducting these analyses. There might be low
sampling for dengue in resource-restrictive settings coupled with under reporting or passive
response to known outbreaks. Furthermore, these samples fail to capture the role of the vector in
these transmission dynamics or other possible hosts for the virus. Despite these restrictions, this
tree demonstrates the circulation of multiple serotypes in distinct geographic regions that might
help assess the risk of severe disease, especially since it is known that DHF is more prevalent
among those infected with DENV-3 and successive infections with multiple serotypes increases
the risk of developing more severe forms of the disease.

7. Kellie Watkins Disease Modeling – Dengue Virus 2 May 2016
http://www.nature.com/scitable/topicpage/dengue-viruses-22400925