Development of a Standardized Sanger Based Method for Partial Sequencing and Genotyping of Dengue Viruses

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Development of a Standardized Sanger-Based Method for

Partial Sequencing and Genotyping of Dengue Viruses

Gilberto A. Santiago,

a

_{Glenda L. González,}

a

_{Fabiola Cruz-López,}

a

_{Jorge L. Muñoz-Jordan}

a

a_{Centers for Disease Control and Prevention, National Centers for Emerging and Zoonotic Infectious Diseases, Division of Vector Borne Diseases, Dengue Branch, San} Juan, Puerto Rico

ABSTRACT

The global expansion of dengue viruses (DENV-1 to DENV-4) has

con-tributed to the divergence, transmission, and establishment of genetic lineages of

epidemiological concern; however, tracking the phylogenetic relationships of these

virus is not always possible due to the inability of standardized sequencing

proce-dures in resource-limited public health laboratories. Consequently, public genomic

data banks contain inadequate representation of geographical regions and historical

periods. In order to improve detection of the DENV-1 to DENV-4 lineages, we report

the development of a serotype-speciﬁc Sanger-based method standardized to

se-quence DENV-1 to DENV-4 directly from clinical samples using universal primers that

detect most DENV genotypes. The resulting envelope protein coding sequences are

analyzed for genotyping with phylogenetic methods. We evaluated the performance

of this method by detecting, amplifying, and sequencing 54 contemporary DENV

isolates, including 29 clinical samples, representing a variety of genotypes of

epide-miological importance and global presence. All specimens were sequenced

success-fully and phylogenetic reconstructions resulted in the expected genotype

classiﬁca-tion. To further improve genomic surveillance in regions where dengue is endemic,

this method was transferred to 16 public health laboratories in 13 Latin American

countries, to date. Our objective is to provide an accessible method that facilitates

the integration of genomics with dengue surveillance.

KEYWORDS

E gene, Sanger, dengue, dengue virus, genotyping, sequencing

D

engue virus, a member of the

Flaviviridae

RNA virus family, consists of four distinct

serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), all of which have been

trans-mitted in more than 100 countries. Approximately 390 million infections have

esti-mated to occur annually (1, 2). Over the last 4 decades, the range of endemic

transmission has increased signiﬁcantly, as dengue is frequently introduced into

re-gions previously unexposed to the virus, often causing epidemics (3–5), and

conse-quently placing approximately 40% of the world population at risk (1, 6). This global

spread, in addition to the characteristic high rate of mutation by ﬂavivirus polymerases,

has contributed to important evolutionary events that affect disease severity and

epidemiology, such as the divergence of subserotypic genotypes and taxonomic

lineages. Many genotypes of epidemiological relevance have been associated with

transmission in a particular geographic region (7–9), disease severity (10–12), or even

increased epidemic potential (13–16). Recent outbreak investigations have reported

molecular evidence suggesting that the introduction of genetically distinct virus strains

may lead to the emergence of new lineages directly related to epidemic transmission

(5, 17). In addition, phylogenetic and molecular epidemiological studies have identiﬁed

the formation, expansion, decline, and extinction of genotypes and subgenotypic

lineages during periods of endemic and epidemic dengue transmission (18–20);

nev-CitationSantiago GA, González GL, Cruz-López

F, Muñoz-Jordan JL. 2019. Development of a standardized Sanger-based method for partial sequencing and genotyping of dengue viruses. J Clin Microbiol 57:e01957-18.https://doi.org/ 10.1128/JCM.01957-18.

EditorYi-Wei Tang, Memorial Sloan Kettering

Cancer Center

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Address correspondence to Jorge L. Muñoz-Jordan, [email protected].

G.A.S. and G.L.G. contributed equally to this work.

Received10 December 2018

Returned for modiﬁcation26 December

2018

Accepted22 January 2019

Accepted manuscript posted online13

February 2019

Published

crossm

28 March 2019

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ertheless, the drivers of these dynamics and their impact on the epidemiology of

disease are not well understood.

Even after decades of studying dengue molecular epidemiology and the modern

advances in genomics, the capacity remains inaccessible for many public health

laboratories running dengue surveillance in regions of endemicity. Although there is a

wide array of published DENV sequencing methods, signiﬁcant limitations have

im-paired implementation, such as high variability between methods, cost, lack of

stan-dardization with clinical specimens, inadequate method evaluation, and high protocol

complexity, which often includes ampliﬁcation of the virus by tissue culture. Only a

limited number of laboratories run some sort of genomic surveillance and have the

capacity to track the local trafﬁc of circulating dengue strains using a diversity of partial

sequencing methods (21–25). However, this function is not frequently performed or

applied to complement surveillance. This is reﬂected in the public repository of dengue

genome sequences, GenBank, which currently holds an unbalanced collection of DENV

genomic data that limits phylogenetic studies. Although the GenBank collection

in-cludes abundant DENV envelope glycoprotein coding sequence (E) data, as historically

targeted for DENV genotyping, this collection is handicapped by the abundance of

redundant sequences and the misrepresentation of certain time periods and endemic

geographical locations (Virus Pathogen Resource,

https://www.viprbrc.org/brc/home

.spg?decorator

⫽

ﬂavi_dengue

).

To increase surveillance and research laboratory capacity and support genomic

surveillance of DENV, we developed the CDC DENV E gene sequencing assay, a

standardized, serotype-speciﬁc, Sanger-based method for genotyping analyses by

se-quencing the envelope glycoprotein coding sequence (E) directly from clinical serum

specimens. We developed this assay for broad implementation in surveillance

labora-tories, including an open-platform protocol that incorporates simple, cost-effective, and

traditional molecular biology techniques to facilitate adaptability. Our assay includes

DNA primers speciﬁcally tailored and validated to detect a wide variety of genotypes of

epidemiological relevance and contemporary circulation globally. The E protein coding

sequence was selected because it provides a convenient target considering the high

abundance of E sequences published in GenBank; in addition to reports that validate E

for genotyping analyses, this fragment contains sufﬁcient phylogenetic signal to

dis-tinguish DENV from other ﬂaviviruses and between serotypes, genotypes, and

individ-ual strains (26–28). Furthermore, DENV phylogenetic tree reconstructions based on

E coding sequence render tree topologies similar to those based on complete

genome sequences facilitating genotyping at a signiﬁcantly reduced cost and effort

(8, 26, 29, 30).

This method aims to facilitate DENV genomic surveillance, improve genotyping, and

foster molecular epidemiology by providing laboratories with a convenient,

cost-effective, and highly efﬁcient tool for sequencing DENV circulating globally directly

from clinical specimens.

MATERIALS AND METHODS

Sequencing target selection and primer design. The DENV E protein coding sequence was selected as the target for amplification and sequencing considering that the length of this sequence, approximately 1,485 bp, contains sufficient conserved sequence regions to differentiate DENV from other flaviviruses and sufficient variability to further classify between serotypes, genotypes, and individual strains (8, 31); it is thus a historically preferred genome fragment for genotyping. The E coding sequence has been the target for genotyping DENV, so more E coding sequences are available than for any other gene in GenBank. These E coding sequences allow for wider sequence sampling for assay design and phylogenetic analysis. All amplification and sequencing primers were designed from multiple sequence alignments of each serotype considering approximately 300 sequences obtained from GenBank and comparing complementarity with 70% and 90% consensus sequences. Each alignment included repre-sentation of all clinically relevant genotypes, global geographical reprerepre-sentation, and sequences isolated between 1990 to 2017. Each alignment was revised to exclude extensive geotemporal redundancy and sylvatic lineages. Up to three noncontiguous mismatches were considered acceptable if sequence changes did not affect melting point temperatures (Tm) significantly; however, we recognize thatin silicomatches may not predict efficient primer binding accurately. The final primer sets for RT-PCR are listed in Table 1, and the sequencing primers are listed in Table 2.

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Viral strains and clinical samples.A panel of 25 presequenced DENV international isolates was obtained from the Centers for Disease Control and Prevention (CDC) Dengue Branch archived collection (Table 3). These 25 strains represent the four serotypes and genotypes of contemporary circulation globally. Sylvatic strains were not included due to the sporadic clinical relevance and the high sequence divergence from the urban-endemic strains, which results in an average of four mismatches per reverse transcription-PCR (RT-PCR) or sequencing primer and reduces the probability of amplification or se-quencing. Each isolate was amplified by tissue culture on Aedes albopictus-derived C6/36 cells, as previously described (32). In addition, a panel of 29 clinical serum specimens RT-PCR positive for all DENV serotypes was obtained from the reference specimen archive of the CDC Dengue Branch collected as part of the island-wide passive surveillance system in collaboration with the Puerto Rico Department of Health and from a dengue outbreak in American Samoa investigated by the CDC Dengue Branch in 2017 (Table 4). The CDC Institutional Review Board (IRB) authorized the use of deidentified samples for this study,

TABLE 1RT-PCR oligonucleotides for DENV E gene ampliﬁcation

Serotype and primer Sequence

DENV-1 PCR-F AGC ACA TGC YAT AGG AAC ATC C DENV-1 PCR-R GCT GAT CGA ATT CCA CAC ACR CC DENV-2 PCR-F TCG CTC CTT CAA TGA CAA TGC DENV-2 PCR-R CAG CTC ACA ACG CAA CCA CTA DENV-3 PCR-F TAG CCC TAT TTC TTG CCC ATT AC DENV-3 PCR-R AGT TCA TTG GCT ATT TGC TTC CA DENV-4 PCR-F AAC AGG RAT CCA GCG AAC TGT DENV-4 PCR-R CTC GCT GGG GAC TCT GGT TG

TABLE 2DENV E gene sequencing primers

Serotype and primer Sequence

DENV-1

D1SEQ1 GCY ATA GGA ACA TCC ATC ACY CAG D1SEQ2 ACG TGT GCY AAG TTY AAG TGT GT D1SEQ3 AAG ACA GCT CAT GCA AAG AAR CAG D1SEQ4 TTG GTG AGA GYT ACA TCG TGR TAG D1SEQ5 GCT GAT CGA ATT CCA CAC ACR C D1SEQ6 CTG GTG TAC CAA TTT TCC CAC AG D1SEQ7 TGG GTC TCA GCC ACT TCY TTC T D1SEQ8 AGC CCT GTT CTA GGT GAG CAA TC DENV-2

D2SEQ1 TYG CTC CTT CAA TGA CAA TGC G D2SEQ2 ACA TGC AAA AAG AAC ATG GAA GGA D2SEQ3 AAT CCC CAY GCV AAG AAA CAG GAT D2SEQ4 CCA TTC GGR GAC AGC TAC ATC AT D2SEQ5 CAG CTC ACA ACG CAA CCA CTA D2SEQ6 TGA TGA TGT AGC TGT CTC CGA ATG D2SEQ7 CTG TGA GTG CCG TGT GCA TG D2SEQ8 CAA RTT TTC TGG TTG CAC GAC T DENV-3

D3SEQ1 AGG CAC TTC CTT GAC CCA GAA D3SEQ2 TGG CAA GGG AAG CTT GGT AA D3SEQ3 AAC AGG AAG GAG CTT CTT GTG ACA D3SEQ4 GAG CCT GTC AAT ATT GAG GCT GA D3SEQ5 TGT CAT GGA TGG GGT GAC CA D3SEQ6 TCC CTC TAT TGG TTC CAG GCA D3SEQ7 CTT GCG ATC CAA GGA CAA CTA CT D3SEQ8 GCG TTG TCT CCA ATT CCA ATY ACT DENV-4

D4SEQ1 TGT CCT AAT GAT GCT GGT CGC D4SEQ2 GAG GAG TTG TGA CAT GTG C D4SEQ3 ATG GTG ACA TTT AAG GTT CCT CAT D4SEQ4 GCT GAG AAT ACC AAC AGT GTA ACC A D4SEQ5 CTC GCT GGG GAC TCT GGT TG D4SEQ6 GCC ATD CGY TTT GCA CCT CT D4SEQ7 TTG AGA TGT CCT GCA AAC ATG T D4SEQ8 ATG GTG ACC TGG GRG TTA TCG TC

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protocol 6874. Serotypes and the relative RNA concentration (threshold cycle [CT]ⱕ30) were conﬁrmed by real-time RT-PCR (33).

Viral RNA extraction.For our evaluation of the CDC DENV E gene sequencing assay, we extracted viral RNA from clinical specimens (serum) or tissue culture supernatant using a QIAamp viral RNA minikit (Qiagen, catalog no. 52904) or other automated method, including the MagNA Pure LC 2.0 instrument (Roche, catalog no. 05197686001) or the MagNA Pure 96 instrument (Roche, catalog no. 05195322001), according to the manufacturer’s recommended protocols.

E coding sequence amplification.E coding sequence was amplified by endpoint RT-PCR using a SuperScript III Platinum one-step RT-PCR system (Invitrogen, catalog no. 12574026) and one set of serotype-specific amplification primers (Table 1). These primers were designed to detect DENV geno-types of contemporary circulation globally and amplify the complete E coding sequence flanked by a small portion of the premembrane (preM) and nonstructural protein 1 (NS1) coding sequences. RT-PCRs were assembled by mixing 13.0␮l of nuclease-free PCR-grade water, 25.0␮l of 2⫻SuperScript reaction mix, 20.0 ␮M concentrations of each DENV forward and reverse primer, 2.0 ␮l of SuperScript III RT/platinumTaqmix, and 8.0␮l of template RNA elution to a final reaction volume of 50.0␮l. RT-PCR analysis was performed in a conventional thermal cycler (SimpliAmp; Thermo Fisher) programmed as follows: reverse transcription (RT) at 55°C for 30 min and 94°C for 2 min; followed by 40 cycles of 94°C for 15 s, 55°C for 30 s, and 68°C for 2 min; and then a final extension at 68°C for 5 min. RT-PCR products were detected and visualized by agarose gel electrophoresis. A 0.8% UltraPure agarose gel (Invitrogen, catalog no. 16500500) was prepared in 1⫻Bionic buffer (Sigma-Aldrich, catalog no. B6186-4L) stained with SYBR Safe DNA stain (Invitrogen, catalog no. S33102) with wells sufficiently thick to contain the complete RT-PCR and loading buffer (approximately 55.0␮l). A 1-kb ladder DNA marker was used to determine the amplicon size. The RT-PCR amplicon length will vary by serotype: approximately 1,700 bp for DENV-1, 1,500 bp for DENV-2, 1,800 bp for DENV-3, and 1,600 bp for DENV-4. This RT-PCR procedure was tested with serial dilutions of reference strains of the four DENV serotypes and dilutions with a real-time RT-PCR CTvalue of⬍30 produced a band with sufficient DNA for successful sequencing (33). Reagents for RT-PCR and electrophoresis can be substituted for other compatible reagents (with the exception of the primers) of similar performance, if available.

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RT-PCR amplicon purification.RT-PCR amplicon bands should be of the expected size with a brightness equal to or greater than the bands in the 1-kb DNA ladder marker. The amplicon bands were excised with a clean scalpel under low-intensity UV light, eliminating as much agarose as possible. The excised slice of gel was placed in a 1.7-ml nuclease-free microcentrifuge tube. DNA from the gel was extracted and purified using a QIAquick gel extraction kit (Qiagen, catalog no. 28706) with the following modifications to the manufacturer’s recommended protocol: the microcentrifuge tube containing the gel slice was filled to capacity with QG buffer and incubated at 50°C for 10 min with frequent vortexing until the gel was completely melted and dissolved in the buffer. Isopropanol was not added to the tube. Half

TABLE 3Panel of sequenced virus isolates

Serotype Strain Genotype GenBank no. Contig size (bp)a

DENV-1 BR/DB001/2003 American-African JF804014 1,667 MX/DB006/2007 American-African JF804019 1,726 PH/DB007/2004 South Pacific JF804020 1,729 PR/DB008/1998 American-African JF804021 1,622 TH/DB047/2006 Asian JF812097 1,738 TI/DB011/2001 South Pacific JF804024 1,732 DENV-2 BR/DB015/2006 American-Asian JF804028 1,529 CI/DB016/2007 American-Asian JF804029 1,504 VN/DB014/1988 Asian I JF804027 1,499 DR/DB018/2003 American-Asian JF804031 1,521 CR/DB017/2003 American-Asian JF804030 1,503 IQT2133 American AY577439 1,525 1349 Cosmopolitan EU056810 1,522 GU/DB019/2001 Asian II JF804032 1,529 IN/DB020/2006 Cosmopolitan JF804033 1,531 DENV-3 BR/DB028/2003 Indian JF804041 1,780 SA/DB028/1995 South Pacific JF804049 1,777 TH/DB055/2006 Thailand JF812105 1,777 EC/DB032/2000 Indian JF804045 1,775 CK/DB030/1991 South Pacific JF804043 1,772 DENV-4 CI/DB039/2006 Indonesian JF804052 1,648 MX/DB042/2006 Indonesian JF804055 1,647 SC/DB045/1994 Indonesian JF804058 1,666 EC/DB041/1999 Indonesian JF804054 1,616 TH/DB060/2006 Southeast Asian JF812110 1,625

a_{Contig size was determined using SeqMan Pro (DNASTAR Lasergene 14).}

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of the contents of the tube was transferred to the Qiagen spin columns by pipetting, and the columns were centrifuged for 1 min at 14,000 rpm. The flowthrough was discarded, and the previous step was repeated until the entire volume of the samples passed through the column. The columns were then washed with 800 ␮l of QG buffer and centrifuged for 1 min at 14,000 rpm. The manufacturer’s recommended protocol continues at this point. Alternatively, the amplicon can be purified using enzymatic methods such as ExoSAP-IT PCR product cleanup reagent (Thermo-Fisher, catalog no. 78201.1.ML). The purified products were quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific).

Sanger sequencing.Sequencing reactions were assembled using a BigDye Terminator v3.1 cycle sequencing kit (Thermo-Fisher, catalog no. 4337455). Optimal purified product input was determined experimentally to range between 40 and 60 ng/reaction. Eight sequencing reactions were performed per sample using serotype-specific primer sets: four primers that generate tiled, overlapping sequences from 5=to 3=and four primers that generate overlapping sequence from 3=to 5=for a minimum 2⫻coverage across the E coding sequence (Table 2). Sequencing reactions were assembled according to the manufacturer’s recommended protocol: 8.0␮l of BigDye Terminator 3.1 Ready Reaction mix, a 3.3␮M concentration of the corresponding single sequencing primer, 60.0 ng of purified DNA product and nuclease-free PCR-grade water to complete a final reaction volume of 20.0␮l. Sequencing reactions were placed on a conventional thermocycler programmed with the following protocol: denaturation at 96°C for 1 min, followed by 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. The ramp rate was adjusted to 1°C/s at each step. Variations of the protocol for the BigDye sequencing reactions should be evaluated and adjusted for optimal results considering the sequencing instrument available and labo-ratory experience. Variations of the protocol may include dilution of the BigDye Terminator 3.1 Ready Reaction mix with 5⫻sequencing buffer and DNA input. BigDye reactions were purified using a BigDye XTerminator purification kit (Thermo-Fisher, catalog no. 4376484) to remove unincorporated dyes following the manufacturer’s recommended protocol. Alternatively, sequencing reactions can be purified by spin column methods like DyeEx 2.0 spin kit (Qiagen, catalog no. 63204). Sequencing was performed on an ABI 3130 XL genetic analyzer (Thermo-Fisher, catalog no. 4359571).

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Trace sequence analysis and assembly. Sequence trace files were analyzed individually using BioEdit software (www.mbio.ncsu.edu/BioEdit/bioedit.html) or Chromas 2.6.5 software (Technelysium). A minimum of 400 bp of high-quality sequence (determined by size of fluorescence signal and clarity of the peak) are expected per trace file. Contig assembly was performed with the eight trace files per sample using the SeqMan Pro program found in the DNASTAR Lasergene 14 software package. The software reports the number of contigs assembled and the number of trace files used to assemble the contigs and

TABLE 4Panel of clinical specimens sequenced

Serotype Sequence label Origin Collection date (yr) GenBank no.

DENV-1 US/DB224/2013 Puerto Rico 2013 MK039554

US/DB225/2013 Puerto Rico 2013 MK039555

AS/DB243/2017 American Samoa 2017 MK244386

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displays the trace file alignment to assemble the contig consensus sequence. Initially, we confirm that we obtained a minimum of 2⫻ coverage across the length of the E coding sequence, followed by a base-by-base curation of the sequence data to guarantee sequence quality. If an ambiguous base is called in the contig consensus sequence, the trace files are analyzed at the specific sequence position to determine the correct base. Once the contig is assembled and the sequence is curated, the consensus sequence is analyzed by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm sequence identity and retrieve similar sequences. The top 5 to 10 sequences from the BLAST results were selected and aligned with our sample consensus sequence using Clustal W or MUSCLE in MEGA 7 software (www .megasoftware.net) to determine consensus sequence orientation and trim sequence data flanking the E coding sequence. This step also serves to identify additional sequences of high similarity that can be used in downstream phylogenetic analyses and provides a general idea of the genotype, as well as the geographic-temporal origin of the query sequence. The resulting DNA sequence was translated to amino acid sequence and compared to similar annotated amino acid sequences retrieved from GenBank. We also confirmed the absence of internal stop codons that might have been introduced by sequencing errors. There are numerous software packages that perform these functions, and we defer software selection to the user considering availability and computational capacity.

Phylogenetic analysis.The sequences obtained with this method were grouped by serotype and aligned with other genotype reference sequences obtained from GenBank (see Table S1 in the supplemental material) using the MUSCLE module in MEGA 7. The multiple sequence alignments were trimmed to the length of the shortest sequence (1,480 to 1,485 bp). Phylogenetic relationships were inferred using the maximum likelihood method based on the Tamura-Nei model and topolo-gies tested with 1,000 bootstrap replicates using IQ-TREE software (http://www.iqtree.org/). Geno-types are marked by brackets and labeled accordingly (7).

Data availability.Sequence data were deposited in GenBank under the accession numbers listed in Table 4.

RESULTS

DENV E coding sequence ampliﬁcation was successful in the 54 clinical serum and

tissue culture samples evaluated (Tables 3 and 4). Amplicons containing E coding

sequence ﬂanked by preM and NS1 coding sequence fragments were sequenced with

the CDC DENV E gene sequencing assay. The assay produced the eight expected

sequence trace ﬁles with a minimum of 400 bp of high-quality data for 29 of 29 clinical

samples and 24 of the 25 virus isolates, which assembled single contigs of more than

1,500 bp with a minimum of 2

⫻

coverage across the E coding sequence segment (Table

3). The overlapping sequencing reactions and the bidirectional sequencing strategy

increased the probability of covering the coding sequence with high-quality sequence

data and facilitated base-to-base calling. For DENV-4 isolate Ecuador 1999, GenBank

accession number

JF804054

, the assay produced seven of the eight sequence trace ﬁles

with the expected minimal size of 400 bp in length; however, a single contig of 1,438 bp

was assembled with 2

⫻

coverage across 80% of the E coding sequence fragment. A

multiple sequence alignment with 10 similar sequences showed that the missing

sequence data were at the 3

=

end of the E coding sequence. To address this issue, we

resequenced this isolate and succeeded obtaining all eight sequence trace ﬁles

assem-bling a contig of 1,616 bp. A highly conserved region of the E coding sequence across

similar sequences was evaluated. Maximum-likelihood phylogenetic trees were inferred

for each DENV serotype using the E coding sequences obtained from this method in

addition to other E coding sequences considered as references for each genotype (Fig.

1). Each of the 54 E coding sequences obtained grouped with the expected genotypes

and relatedness to genotype reference sequences were supported by bootstrap scores

of higher than 75%. In addition, our genotyping results concurred with the results

obtained from a recently developed web-based online Genome Detective Dengue Virus

Typing Tool for dengue virus genotyping (

https://www.genomedetective.com/app/

typingtool/dengue/introduction

) which aligns any DENV-1 to DENV-4 sequence with

preselected genotype reference sequences and outputs a phylogenetic tree.

DISCUSSION

The transmission of DENV continues to expand globally, increasing the number of

regions exposed and people at risk of infection. Real-time monitoring of transmission

and epidemics is necessary to forecast further virus spread, which will impact the

design of preventive interventions and treatment measures. Several studies have

shown how, occasionally, foreign virus introductions or even endemic transmission can

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FIG 1Genotyping DENV using E gene sequence. Phylogenetic reconstruction was used to determine DENV genotypes by the maximum-likelihood method based on the Tamura-Nei model of nucleotide substitution. (A) DENV-1; (B) DENV-2; (C) DENV-3; (D) DENV-4. Asterisk-labeled taxa indicate sequences obtained by this method. Due to the sequence similarity and space limitations, only two of the ten DENV-2 American Samoa 2017 sequences were included in the maximum-likelihood tree.

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FIG 1(Continued)

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generate dengue outbreaks regardless of previous exposure to the virus (5, 17, 34). This

supports the importance of genomic surveillance to understand the epidemiology of

dengue in a region. The CDC DENV E gene sequencing assay was developed to provide

a simple and convenient standardized tool for DENV partial sequencing, for

genotyp-ing, and to harmonize genomic surveillance of dengue viruses. Such a tool will be

readily adaptable to public health laboratories and provide the opportunity to monitor

genetic changes associated with circulating genotypes or other epidemiological

fac-tors. The selection of the E coding sequence as sequencing target provides sufﬁcient

data to perform phylogenetic inferences for genotyping and lineage classiﬁcation with

tree topologies that closely resemble those obtained from complete genome

se-quences (7, 26, 35).

This assay was developed to sequence directly from diagnostic serum specimens

using a simple approach to amplify the target region and obtain reliable sequence data

taking advantage of the Sanger sequencing method. Despite the advantages that

next-generation sequencing technologies offer, the Sanger method is more accessible

to laboratories in regions where dengue is endemic and provides a nimble strategy to

process smaller numbers of samples with small genomes with high accuracy. Our

evaluation of this assay demonstrated the capacity to sequence a wide variety of

international DENV genotypes within each serotype and with wide sequence variation.

Only on one occasion was a DENV-4 isolate not sequenced entirely, with a contig

formed by seven sequence trace ﬁles, and we speculate that performance of the eighth

sequencing reaction could have been affected by insufﬁcient loading of amplicon

product into the reaction. However, this was corrected by resequencing the isolate with

the proper concentration of DNA template. A simple genomic analysis demonstrated

that the sequences obtained by this method readily apply to phylogenetic studies

relevant to molecular and genomic surveillance such as genotype detection. Although

whole-genome sequences are more suitable for evolutionary studies and provide

higher phylogenetic resolution for lineage classiﬁcation, the E coding region has been

a more popular target historically; hence, there is a larger availability and

representa-tion in public databases. Consequently, the accuracy of all phylogenetic analyses based

on this or any other sequencing method is restricted by the sequence sampling and

availability in public domain. Because all national laboratories in the Americas are

currently running the same RT-PCR procedures for DENV-1 to DENV-4 detection (33),

the RNA extracts already obtained can be readily sequenced using this procedure.

Therefore, we have deployed this procedure in collaboration with the Pan American

Health Organization, the National Health Institute of Surveillance and Reference in

Mexico, and the Institute Fiocruz in Brazil. These efforts have made it possible to

incorporate genomic surveillance components in 16 public health laboratories in 13

countries in the region.

The success rate of this assay when used directly on diagnostic serum specimens

is limited by the sample quality, i.e., maintenance of the cold chain, the time of

specimen collection (i.e., acute stage 0 to 5 days after onset of symptoms), and the

viral RNA concentration (i.e.,

C

T

⬍

30 determined by real-time RT-PCR, although a

low

C

T

value may not reﬂect the integrity of the RNA genome molecule). Further

in

silico

analyses of the primer sequences showed several mismatches with modern

sequences of Asian origin. To address this, we offer a list of the primers with IUPAC

nucleic acid codes in positions where mismatches were detected (Table S2);

how-ever, due to the unavailability of Asian dengue virus isolates in our laboratory, we

were unable to validate these primer sequences with PCR testing. For further

evolutionary and molecular epidemiological studies, we recommend methods that

provide complete genome sequence.

In conclusion, surveillance and research laboratories can use this method to

collect dengue genomic information to strengthen the understanding of DENV

genotypes, diversity, and transmission dynamics and to improve disease prevention

strategies.

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at

https://doi.org/10.1128/JCM

.01957-18

.

SUPPLEMENTAL FILE 1

, XLSX ﬁle, 0.01 MB.

ACKNOWLEDGMENT

G.A.S. is a research scientist at the Centers for Disease Control and Prevention,

Dengue Branch, San Juan, Puerto Rico. His research is focused on the development

of molecular diagnostic tests and the study of the molecular epidemiology of

arboviruses.

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