Molecular Epidemiology of a Type 4 Dengue Virus Outbreak in Paraguay, 2019-2020 Open Access

Shen, John (Spring 2022)

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Background: Dengue virus type 4 (DENV-4), first detected in Paraguay in 2012, had persisted in the country as a non-predominant type. In 2019-2020, DENV-4 became predominant and caused an outbreak in Paraguay that spanned two waves: a smaller first wave in mid-2019 and a larger second wave between late 2019 and early 2020. We investigated the molecular epidemiology of the DENV-4 outbreak to understand its origin and evolution.


Methods: Patients with suspected dengue were enrolled in a cross-sectional study between January 2019 and March 2020 from two clinical facilities in Asunción, Paraguay. We selected DENV-4 positive samples for complete viral genome sequencing and phylogenetic analysis. Epidemiologic metadata were collected through a survey. Logistic regression models were run to find potential single nucleotide polymorphism (SNP) associations with disease severity.


Results: We sequenced complete DENV-4 genomes from 61 patients, largely concentrated in the Greater Asunción metropolitan area. Phylogenetic analysis revealed that outbreak viruses from 2019-2020 belonged to genotype II. The samples sequenced in this study were closely related to a small number of DENV-4 viruses detected in Paraguay in 2018, differing by only two synonymous mutations in NS3 and NS5. The shared ancestor between our Paraguay sequences and the closest sequence outside of Paraguay (Brazil 2013) dated to October 2010 (95% HPD: Mar 2010-May 2011). Among the outbreak sequences, we observed two clades, which diverged from a common ancestor in August 2017 (95% HPD: May 2017-Nov 2017), differed by three nucleotides (synonymous mutations in the E, NS3, and NS5 proteins), and were not separated by time or geography. We did not find a significant difference in the percentage of severe dengue cases between the clades (p=0.70), nor any SNPs associated with dengue severity.


Conclusions: Overall, our results suggest that a lineage of DENV-4 may have been circulating undetected in South America since 2010. We did not find evidence for substantial viral genomic differences between outbreak and pre-outbreak DENV-4 sequences, or between the first and second wave. A larger study with unbiased DENV sampling may determine if observed SNPs affect clinical manifestation of dengue.

Table of Contents

Introduction. 1

Materials and Methods. 2

Ethical considerations. 3

Study samples. 3

Sequencing. 3

Selection of reference sequences. 4

Phylogenetic analysis. 5

Analysis of SNP effect on dengue severity. 6

Results. 7

Descriptive analysis of severe and non-severe dengue groups. 7

Case distribution by symptom onset 7

Case distribution by geography. 8

Maximum likelihood tree. 8

Temporal structure of DENV-4 evolution. 9

Maximum clade credibility tree. 9

SNP effect on dengue severity. 11

Discussion. 11

Conclusions. 14

References. 15

Figures and Tables 18

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