The evolutionary dynamics of DENV 4 genotype I over a 60-year period.

Dengue virus serotype 4 (DENV 4) has had a relatively low prevalence worldwide for decades; however, likely due to data paucity, no study has investigated the epidemiology and evolutionary dynamics of DENV 4 genotype I (DENV 4-I). This study aims to understand the diversity, epidemiology and dynamics of DENV 4-I. We collected 404 full length DENV4-1 envelope (E) gene sequences from 14 countries using two sources: Yunnan Province in China (15 strains during 2013-2016) and GenBank (489 strains up to 2018-01-11). Conducting phylogenetic and phylogeographical analyses, we estimated the virus spread, population dynamics, and selection pressures using different statistical analysis methods (substitution saturation, likelihood mapping, Bayesian coalescent inference, and maximum likelihood estimation). Our results show that during the last 60 years (1956-2016), DENV 4-I was present in mainland and maritime Southeast Asia, the Indian subcontinent, the southern provinces of China, parts of Brazil and Australia. The recent spread of DENV 4-I likely originated in the Philippines and later spread to Thailand. From Thailand, it spread to adjacent countries and eventually the Indian subcontinent. Apparently diverging around years 1957, 1963, 1976 and 1990, the different Clades (Clade I-V) were defined. The mean overall evolution rate of DENV 4-I was 9.74 (95% HPD: 8.68-10.82) x 10(-4) nucleotide substitutions/site/year. The most recent common ancestor for DENV 4-I traces back to 1956. While the demographic history of DENV 4-I fluctuated, peaks appeared around 1982 and 2006. While purifying selection dominated the majority of E-gene evolution of DENV 4-I, positive selection characterized Clade III (Vietnam). DENV 4-I evolved in situ in Southeast Asia and the Indian subcontinent. Thailand and Indian acted as the main and secondary virus distribution hubs globally and regionally. Our phylogenetic analysis highlights the need for strengthened regional cooperation on surveillance and sharing of sample sequences to improve global dengue control and cross-border transmission prevention efforts. Author summary Dengue virus (DENV) can be classified into four serotypes, DENV 1, 2, 3 and 4. Although DENV 4 is the first dengue serotype to diverge in phylogenetic analyses of the genus Flavivirus, this serotype occurs at a low prevalence worldwide and spreads the least rapidly. Similar to other serotypes, DENV 4 can also cause severe dengue (SD) disease manifestations, such as dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). To date, no study has investigated the epidemiology and dynamics of DENV 4 genotype I comprehensively. In this study, we seek to address this gap. Our study shows that the distribution of DENV 4-I is mainly restricted to Southeast Asia and the Indian subcontinent. The most recent spread of DENV 4-I likely originated from Southeast Asia-initially circulating in the Philippines, then Thailand and later on the Indian subcontinent. Viruses evolved in situ in Southeast Asia and the Indian subcontinent, respectively. Although DENV 4-I occasionally spread elsewhere, this genotype did not become widely established. The overall evolution rate of DENV 4-I was comparable with that of DENV 2-4. The nucleotide sequences indicates that the demographic history of DENV 4-I fluctuated with peaks apparent during parts of the 1980s and 2000s. Although a weak positive selection existed in Clade III -predominately in Vietnam, purifying selection dominated the E-gene evolution of DENV 4-I.