Emergence And Circulation of Dengue Virus Serotype 2 In Guangzhou Over A Period of 20 Years

Objectives The dengue epidemic in Guangzhou has imposed a rising burden on society and health infrastructure. Here we present the genotype data for dengue virus serotype 2 (DENV-2) to improve the understanding of dengue epidemic. Methods We sequenced the envelope gene of DENV-2 obtained from patient serum sample, and subsequently performed the maximum-likelihood phylogenetic analysis using PhyMLv3.1, the maximum clade credibility analysis using BEAST v.1.10.4 and selection pressure analysis using Datamonkey 2.0 . Results The DENV-2 prevalent in Guangzhou region related to the strains of Southeast Asian countries.Our results suggest that the Malaysia/Indian subcontinent genotype is prevalent in Guangzhou and no genotype shift has occurred during the last 20 years. Episodic positive selection was detected at one site. Conclusions Prevention and monitoring imported cases are important for local control. The shift between the lineages of the Malaysia/Indian subcontinent genotype, which originated at different time points, may be the underlying cause of rising DENV-2 cases in Guangzhou.The low rate of dengue haemorrhagic fever in Guangzhou may be explained by the dominance of the less virulent Malaysia/Indian subcontinent genotype.


Introduction
Dengue is caused by infection with dengue virus (DENV), which is a member of the genus Flavivirus, family Flaviviridae. Approximately 96 million dengue infections were estimated globally in 2010, of which 70% were in Asia [1].The clinical manifestations of dengue can range from mild fever, known as dengue fever, to the lethal forms of dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Despite extensive efforts, no effective vaccines or drugs against DENV have been developed to date. Therefore, prevention strategies such as preventing further mosquito bites in patients, cleaning vector breeding sites, and using pesticides are the main methods to control dengue at present.
There are four serotypes of DENV, each of which can be divided into several genotypes based on phylogenetic analysis. Dengue virus serotype 2 (DENV-2) is considered to have greater potential to cause DHF/DSS and spread than the other three serotypes. Some studies have shown that secondary infection with DENV-2 after the rst heterotypic infection is more likely to result in DHF/DSS than the secondary infection of the other three serotypes [2].
Since dengue fever was rst recorded in 1978, dengue has been an epidemic in Guangzhou, China for nearly 40 years, imposing an increasing burden over time. Over the past decade, more than 40,000cases of dengue have been reported in the region, dominated by DENV-1 [3,4]. However, DENV-2 is also detected in Guangzhou, which has been associated with outbreaks in some communities over the last two decades. With the spread and epidemic of DENV-2, the risk of a DHF/DSS outbreak is increasing. Furthermore, a speci c genotype may have the propensity to cause DHF/DSS and be transmitted e ciently by vectors [5,6]. Therefore, it is necessary to closely monitor the genetic diversity and genotype variations of DENV-2 to best understand dengue epdemics in Guangzhou.
Toward this end, we analysed serum samples of patients suspected to have dengue that were sent to the Guangzhou Center for Disease Control and Prevention for diagnostic purposes by hospitals from 2001 to 2020. By phylogenetic analysis of the DENV-2 strains presenting during this time, this investigation will provide reference information to scientists and public health o cials dealing with prevention and control of dengue.

Sample collection
Blood samples were obtained from patients with symptoms suggestive of dengue, such as sudden high fever with headache, arthralgia, and/or myalgia, according to the diagnosis for dengue fever promulgated by the National Health Commission of the People's Republic of China [7].

Virus isolation and sequencing
The serum samples were analysed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) on arrival, using a dengue virus RT-qPCR kit (Jiangsu BioPerfectus Technologies Co.,Ltd.,China). Supernatants of cell cultures with no CPE were added to new C6/36 monolayers. Inoculated cells without CPE after three generations were considered to be negative for a DENV isolation.
In 2019 and 2020, to improve the quality or delity of the sequences,RNA extracted from the serum samples was used for RT-qPCR and direct sequencing of the envelope gene without virus isolation, as recommended by Leitmeyer et al. [8]. Therefore, more sequences were acquired in 2019 than in other years.

Phylogenetic and molecular clock analysis
The maximum-likelihood phylogenetic tree of the obtained envelope sequences acquired in Guangzhou along with the reference sequences retrieved from GenBank [9] was constructed using PhyML v3.1. The substitution model was determined by SMART model selection of the Bayesian information criterion [10].
The fast likelihood-based method of eBayes was applied owing to the large number of sequences. Genotypes were grouped according to the criteria de ned by Rico-Hesse [11].
A maximum clade credibility (MCC) tree was constructed for the same sequences using BEAST v.1.10.4. The detected date for each sequence was used as the calibration date. The tree was visualised using Bayesian Evolutionary Analysis Utility (BEAUti) with the following settings: substitution model,GTR; base frequencies, estimated; site heterogeneity model,gamma + invariantsites; number of gamma categories, 4; clock type, uncorrelated relaxed clock; tree model, random starting tree; length of chain, 100,000,000; echo state to screen every 10,000;and log parameters every 10,000. The results of both the maximumlikelihood and MCC phylogenetic trees were visualised and edited using FigTree.

Selection pressure analysis
Episodic adaptive selection was evaluated using the Mixed Effects Model of Evolution (MEME) algorithm implemented by Datamonkey 2.0 [12]. A signature of positive selection for each site can be determined when the β + parameter, representing the rate of nonsynonymous substitutions (dN), is greater than α, representing the rate of synonymous substitutions (dS).
3 Results Table 1  Over the 20 years, 148 DENV-2 envelope gene sequences were obtained from the sera of patients in Guangzhou. All sequences were deposited in GenBank.
The phylogenetic tree shown in Figure 1

Discussion
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 ndings 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][20][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][24][25][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 con rm 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. 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][28][29][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 e cient 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 identi ed 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.

Declarations
Ethics approval and consent to participate: The Ethics Committee of the Guangzhou Centre for Disease Control and Prevention approved the present study.
Written informed consent was obtained from all the participants in the study or from the parent/guardian when the participant was a minor (younger than 18 years old). All procedures involved in this work complied with the ethical standards of the relevant national and institutional committees on human experimentation and with the Declaration of Helsinki (2008 amendment). Consent for publication: Not applicable.
Availability of data and materials: The sequences analysed during the current study are available in the GenBank repository, https://www.ncbi.nlm.nih.gov/genbank/. The data that support the ndings of this study are available from the corresponding author. Maximum-likelihood midpoint rooted phylogenetic tree of DENV-2 envelope sequences derived from 148 sequences detected in Guangzhou and 56 reference sequences retrieved from GenBank. Sequences of Guangzhou are identi ed according to the accession number, isolated year, and lab number. Reference sequences from GenBank (marked with a triangle) are identi ed using the accession number, country, and year. Bootstrap support, with >75%, is shown next to the branches. Different colours present different genotypes.