Over the last century, DENV-2 caused epidemics in 1986, 1987, 1988, and 1993 in Guangzhou[13,14], and has subsided since. Although no infection of DENV-2 was detected prior to 2005, the number of DENV-2 cases continued to increase annually between 2010 and 2018. At the same time, we found that the percentage of domestic cases increased from 80.95% to 95.31% between 2015 and 2018, reaching a peak in both number and percentage in 2018. Although the number and percentage of domestic cases declined slightly in 2019, they were the second highest compared to previous years. These findings indicate that DENV-2 is spreading in Guangzhou. Moreover, there was a sharp decrease in DENV-2 cases and in the number of total DENV cases in 2020, which might be related to the quarantine imposed due to the corona virus disease 2019 outbreak[15]. That is, limiting imported cases with the quarantine helped to restrain the local epidemic, highlighting the relevance of monitoring imported cases for local control.
This epidemiological investigation revealed that 83.72% of imported cases, most of which were returning travellers, originated from Southeast Asian countries. The results of a BLAST search in GenBank and the phylogenetic tree also showed that the DENV-2 strains sequenced in Guangzhou were closely related to the strains in Southeast Asian countries, which was similar to the characteristic of dengue epidemics involving the other three serotypes in Guangzhou[3,16,17]. The World Health Organization statistics revealed that infections in Southeast Asian countries account for half of the global dengue burden. From 2015 to 2019, dengue cases in Southeast Asia increased by 46% (from 451,442 to 658,301)[18]. China is contiguous with Southeast Asian countries, with active economic exchange. With the opening of private travel abroad, the number of travellers to Southeast Asian countries has increased steadily[19–21]. This situation is not unique to China, as the spread of dengue viruses by travellers has become a worldwide problem[22]. Therefore, better preparation is needed with strict regulations to prevent the spread of infection when travelling in endemic areas. For example, a convenient and rapid method for screening viremia that can be applied at customs may help to curb the importation and spread of DENV.
The phylogenetic tree showed that the dominant genotype in Guangzhou was the Malaysia/Indian subcontinent genotype, which comprised 142 of the 148 DENV-2 sequences (95.95%). Moreover, four (66.67%) of the six strains constituting the Southeast Asia genotype were detected among imported cases, indicating that the Malaysia/Indian subcontinent genotype was responsible for the domestic epidemic, and no genotype shift was observed in the 20 years. The most recent common ancestor of all Malaysia/Indian subcontinent strains was estimated to have appeared in 1955. However, when dividing the Malaysia/Indian subcontinent genotype into its different lineages, a shift was observed between 2013 and 2014. Lineage GZ5 has prevailed since 2014, and the number of DENV-2 cases began to rise after that point. Although there is no clear relationship between lineage and virulence in DENV, outbreaks, limited circulation, and spreading related to shifts in lineages have been reported[23–26]. Substitutions in the envelope gene that may result in maturation and activation of macrophages, with consequent enhancement of the immune response characterised by increased production of cytokines, are considered to be the likely cause of the prevailing differences among lineages. Positive selection analysis of the GZ5 lineage by MEME showed signs of directional selection. However, further research is needed to confirm whether the lineage shift is responsible for this rise in cases. Meanwhile within the same year, the strains could also be distributed in different lineages; for example, the sequences from isolates obtained in 2018 were distributed among lineages GZ1, GZ3, GZ4 and GZ5.This co-epidemic of different lineages showed different origins of DENV, which complicates the epidemic situation in Guangzhou.
The MCC tree revealed that the strains of Guangzhou also originated from different time periods. Strains that clustered into lineage GZ5 shared the eldest ancestor in 1995, whereas strains belonging to lineage GZ1 emerged in 2016. The domestic and foreign strains of lineage GZ2 detected in 2014 and 2016 shared the same ancestor in 2012. The strain then evolved during 2014 and 2016 in Guangzhou and spread not only in China but also in Thailand and Vietnam. The evolution and dissemination of DENV-2 were most obvious in this branch.
Secondary infection with DENV-2 after infection with heterotypic DENV is believed to be associated with an increased risk of DHF/DSS[2]. However, with the epidemic of DENV-1 in Guangzhou persisting for more than 20 years and the rising number of DENV-2 cases[3,4], the incidence of DHF remained relatively low compared with the global incidence or the incidence of Southeast Asian countries[27–30]. Some studies have revealed that secondary infection of the American genotype of DENV-2 failed to cause DHF[31],and other extensive studies indicated that the Southeast Asian genotype was more efficient at infection and was also more likely to cause DHF[5,6,31,32]. Of the 148 sequences detected in Guangzhou, only six sequences (4.05%) belonged to the Southeast Asia genotype, four of which were identified from imported cases. This revealed that the epidemic of the Southeast Asia genotype was rare, which may explain the low incidence of DHF in Guangzhou. Our results also suggest that the Malaysia/Indian subcontinent genotype may be less virulent than the Southeast Asian genotype. However, further studies are needed to determine whether the incidence of DHF/DSS is low in other areas with an epidemic dominated by the Malaysia/Indian subcontinent genotype. Determining the critical differences between genotypes and host immune mechanisms that may enhance the pathogenesis of genotypes may provide new insight to better understand the mechanism of DHF/DSS.