Genotype of dengue virus serotype 1 in relation to severe dengue in Guangzhou, China

Guangzhou has been the city most affected by the dengue virus (DENV) in China, with a predominance of DENV serotype 1 (DENV‐1). Viral factors such as dengue serotype and genotype are associated with severe dengue (SD). However, none of the studies have investigated the relationship between DENV‐1 genotypes and SD. To understand the association between DENV‐1 genotypes and SD, the clinical manifestations of patients infected with different genotypes were investigated. A total of 122 patients with confirmed DENV‐1 genotype infection were recruited for this study. The clinical manifestations, laboratory tests, and levels of inflammatory mediator factors were statistically analyzed to investigate the characteristics of clinical manifestations and immune response on the DENV‐1 genotype. In the case of DENV‐1 infection, the incidence of SD with genotype V infection was significantly higher than that with genotype I infection. Meanwhile, patients infected with genotype V were more common in ostealgia and bleeding significantly. In addition, levels of inflammatory mediator factors including IFN‐γ, TNF‐α, IL‐10, and soluble vascular cell adhesion molecule 1 were higher in patients with SD infected with genotype V. Meanwhile, the concentrations of regulated upon activation normal T‐cell expressed and secreted and growth‐related gene alpha were lower in patients with SD infected with genotype V. The higher incidence of SD in patients infected with DENV‐1 genotype V may be attributed to elevated cytokines and adhesion molecules, along with decreased chemokines.

Dengue fever (DF) is an emerging arboviral disease caused by the dengue virus (DENV) and is endemic in tropical and subtropical countries worldwide. 1The incidence of DENV infections was estimated to be almost 400 million per year, of which about 25% were clinically apparent decades ago. 2 The reported cases of dengue have increased dramatically worldwide in recent decades, leading to dengue being identified as one of ten threats to global health in 2019. 35][6][7] The dengue fatality rate ranges from 0.05% to 0.5%, primarily attributed to severe dengue (SD). 8About half of the world's population is now at risk of dengue. 9 China, instances of dengue infection were over 80 0000 between 2005 and 2020, with more than 70% of cases concentrated in Guangdong. 10In addition, Guangzhou has reported the highest cumulative dengue cases in Guangdong over the past 30 years, making it a key city in controlling dengue epidemic in mainland China. 10DENV consists of four serotypes named 1−4 based on antigenic heterogeneity. 11DENV serotype 1 (DENV-1) not only remained the most predominant serotype, 10 but also was the main cause of SD in Guangzhou. 12DENV-1 has five different genotypes named I−V. 13Although all genotypes of DENV-1 have been detected in the past decades, genotype I still predominates in Guangzhou. 10,14ral factors, inflammation, antibody-dependent enhancement (ADE), and endothelial activation are often considered as risk factors for SD. 15 The diversity of DENV serotypes and genotypes may contribute to the increasing number of patients with SD due to ADE. 16 Previous studies have revealed that five genotypes of DENV-1 were present in Guangzhou. 10,17However, the association between the disease severity of dengue and DENV-1 genotype was still unknown.
To understand the relationship between DENV-1 genotype and dengue disease severity, DENVs were isolated from patients' serum for sequencing and genotype identification in this study.

Information on genotype-specific clinical manifestations was used
to analyze the clinical characteristics of dengue patients in relation to DENV-1 genotype, while levels of inflammatory mediator factors were used to investigate the immune response on DENV-1 genotype.

| Study design and participants
This retrospective observational study was based on the Dengue Clinical Research Database of Guangdong Province (DCRDGP).The database was built by 19 hospitals in Guangdong. 12The clinical data used in this study was mainly originated from those hospitals.The patients infected with DENV-1, confirmed by laboratory tests, were recruited for this study.The data of demographic information, clinical manifestations, and laboratory indicators data were collected from the DCRDGP.
The study subjects were diagnosed with SD according to the 2009 dengue guidelines by the World Health Organization (WHO). 18e criteria for SD were followed: (1)

| Diagnostic tests
According to the manufacturer's instructions, RNA was extracted from patients' serum sample respectively using the QIAamp ® Viral RNA Mini Kit (Qiagen).Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) assays were performed to determine specific serotypes by Dengue Detection Kit (Daan).

| Genomic sequencing
The serum samples collected from dengue patients from Day 1−10 of onset were inoculated in C6/36 cells.The DENVs were successfully isolated from serum when cytopathic effects were observed within 7 days, and then they were cultivated in C6/36 cells for up to 5 generations.The serum was abandoned because DENV could not be isolated within three generations.The 200 μL supernatants collected from the virus culture medium were used to extract DENV RNA by QIAamp ® Viral RNA Mini Kit (Qiagen).The extracted RNA was quantified using the Qubit 2.0.RNA integrity was assessed using agarose gel electrophoresis.The purity of RNA was assessed by calculating the ratio of absorbance readings at 260 and 280 nm.Illumina HiSeq.2500 platform was used for high-throughput sequencing (paired-end, 2 × 150 bp).The CLC Genomics Workbench (version 11.0) was used for sequence splicing.Notably, not all serum samples collected from patients with SD contributed to successful DENV isolation.Furthermore, one DENV genome sequence could not be linked after sequencing.
In total, 122 DENV-1 genome sequences were used for the phylogenetic analysis in the study.The phylogenetic tree was constructed based on the entire open reading frame (ORF) genome sequences of DENV-1.Before generating an alignment, a global genetic diversity sequence data set was compiled using the NCBI Virus database (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?SeqType_s=Nucleotide).By using the query "complete" 1988 DENV-1 complete genomic sequences were obtained from NCBI Virus.To enable cross-country analysis, a random data set was obtained by downloading 10 sequences from each country using a stratified sampling method.A total of 466 DENV-1 complete genomic sequences were collected from the random database.After integrating the corresponding DENV sequences from this study into the data set, rapid sequence alignment was performed using MAFFT v7 (https://mafft.cbrc.jp/alignment/server/index.html).Then, visually inspected and refined them in Aliview version 1.26 (https://github.com/AliView/AliView). MEGA 11 (https://www.megasoftware.net/) was used to mask noncoding sequences and remove sequences that did not contain the entire ORF genome.The sequences performed between 2001 and 2022 were recruited for aligned.In total, 257 reference sequences and 122 sample sequences were used to create an aligned database, which was then utilized to construct a maximum likelihood phylogenetic tree using the best-fit substitution model in IQTREE v2.2.2.6, employing a general time reversible substitution model with 1000 bootstrap replicates.The phylogenetic tree was visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/ figtree/).In the figure, 122 genomic sequences of samples and 13 reference sequences were briefly included.

| Measurement of inflammatory mediator factors
The plasma samples were isolated from patients with SD on Day

| Statistical analyses
The cytokine levels in various groups were visualized using GraphPad Prism 8.3 (GraphPad Software, Inc.).Continuous variables are summarized using the median with interquartile range, while categorical variables are summarized using the frequencies and proportions.Continuous variables were compared using the nonparametric Mann-Whitney U test for skewed variables.Categorical variables were assessed using the chisquare test or Fisher's exact test.All the statistical analyses were performed with SPSS Statistics 21.0 software (IBM Corp.).

| Genomic characteristics of DENV-1 in patients hospitalized in Guangzhou
During 2013−2019, DENV-1 RNA was detected in 1589 dengue patients by qRT-PCR.A total of 131 DENV-1 strains were isolated from the serum of hospitalized patients, and an additional 122 genomic sequences were performed (Figure 1).Among the 122 patients who provided serum samples for DENV genome sequencing, 93 patients were diagnosed with DF while 29 patients were diagnosed with SD.
Genotype II and genotype III of DENV-1 were not identified in the patients.

| Clinical manifestation features of dengue in patients infected with different genotype of DENV-1
The main clinical manifestations of the patients infected with DENV-1 are presented in Table 1.A total of 121 patients were divided into genotype I group (84, 69%) and genotype V group (37, 31%).Sixtythree (52.1%) patients were males, and there was no significant difference between the two groups in terms of gender.The median age of the patients was 56 years old.The patients in genotype V were older than those in genotype I (63 vs. 50, p = 0.029).All the patients were adults.Between the two groups, no significant difference was observed in co-morbidities overall (35.7% vs. 37.8%, p = 0.823).

| Classification of SD in both groups
According to the SD criteria of the 2009 WHO guidelines, we classified the patients with SD into two groups (Table 2).Among patients infected with genotype V, SOI was the most common presentation (47%), followed by SH (35%), and SPL (18%).Patients infected with genotype I more prevalent in SH (52%), followed by SOI (42%), while SPL (6%) was a less common manifestation.In addition, no difference of SD presentation was observed in genotype I and genotype V groups.

| Relationship between genotypes of DENV-1 and dengue disease severity
To understand the association between DENV-1 genotype and SD, we confirmed the genotype of DENV-1 in each dengue-infected patient.
The genotype of DENV-1 associated with disease severity was further assessed and is presented in Table 3.In the 29 patients with SD conformed genotype, 15 patients were infected with genotype I and 14 patients infected with genotype V.Among the DENV genome sequences data of 93 patients with DF, 69 cases were clustered to genotype I, 23 to genotype V and 1 to genotype IV.Among the patients with SD, a higher percentage had genotype V infection compared to genotype I infection (37.8% vs. 17.9%,p = 0.018).

| Inflammatory mediator factors between the SD patients with infection of genotype I or genotype V
The levels of inflammatory mediator factors in the final febrile phase and critical phase of the disease were investigated to comprehend the variation in inflammation resolution between patients with SD in the genotype V group and the genotype I group (Figure 3).Comparing to the genotype I group, levels of proinflammatory cytokines such as TNF-α and IFN-γ were significantly higher in the genotype V group (p < 0.05).However, no difference in IFN-γ levels was found between genotype I and healthy individuals (NC).Elevated levels of the antiinflammatory cytokine IL-10 were also observed in genotype V (p < 0.05).Meanwhile, an evident decrease in chemokines, such as RANTES and GRO-α, was observed in genotype V (p < 0.05).In addition, elevated levels of the adhesion molecule sVCAM-1 were significantly detected in the genotype V group compared to the genotype I group (p < 0.05).However, levels of TNF-α, IL-10, RANTES, and GRO-α were significantly decreased in patients with SD compared with the NC group (p < 0.05).
Even the three chemokines, as IL-8, IL-15, and IL-17A, and the chemokine MCP-1, were significantly higher in patients with SD than in the NC group (p < 0.001).However, no differences in the levels of inflammatory mediator factors mentioned above were found between the genotype V group and the genotype I group (Figure 4).
F I G U R E 2 Polygenetic tree of DENV-1 sequences isolated in Guangzhou.The phylogenetic tree based on 122 DENV-1 samples and 13 references were constructed by the maximum likelihood method.The red labels point to the samples collected from patients with severe dengue (SD), the blue labels point to the samples collected from patients with dengue fever (DF), and the black labels point to the references.The sample sequences are named by accession number, country origin and classification of dengue.The reference sequences are named by GenBank accession number, country origin and date of isolation.DENV-1, dengue virus serotype 1.
According to the antigenic features, DENVs are classified into four serotypes.DENV-1 has dominated the recent dengue outbreaks in Guangzhou, 14,[19][20][21] the city bearing the heaviest disease burden of dengue in China. 22Sequence-based serotyping of DENV has revealed the existence of typical clades known as genotypes. 23,24DENV-1 comprises five genotypes named I, II, III, IV, and V, respectively. 4In this study, sequencing of 122 DENV-1 cases found that genotype I (68.9%) was the most predominant in Guangzhou, followed by genotype V (30.3%).It is consistent with the study reported by Jiang, L. Y and his colleagues. 19Previous studies have reported that while DENV-2 infection was extensively associated with SD, 25,26 a higher incidence of SD was observed with DENV-1 infection compared to DENV-2 infection in China and Singapore. 11,27,280][31] To understand the relationship between DENV-1 genotype and clinical manifestations, 122 patients who provided serum samples for sequencing were recruited for this study.Note: χ 2 test was used in the data analysis.
In the present study, we found that SD was significantly associated with cases infected with the DENV-1 genotype V.
Genomic sequencing technology has been applied in primary hospitals since the COVID-19 pandemic, making it easier to determine the genotypes of the four serotypes of DENV nowadays.
3][34][35] The classification of SD presentation was distinguished by dengue guidelines (2009).SH was common in patients infected with genotype I, as revealed in Suppiah J and his colleagues' study. 31rthermore, we found that SOI was pronounced in patients infected with genotype V, followed by SH and SPL.For the first time, the relationship between the genotype of DENV-1 and SD was clearly described.
Laboratory tests are objective indicators of the patients' condition.Moreover, no differences were found in the indicators related to SPL, such as HCT and ALB.Similarly, the indexes of SOI, including ALT, AST, and BUN, and the parameters of SH like PT and INR, showed no significant variances between patients in the two groups.Genotype may not contribute to SOI resulting from DENV-1 infection.Although the presence of SH was no different in cases infected with either genotype I or genotype V, lower PLT was observed in patients infected with genotype V obviously.
The higher proportion of bleeding in cases of genotype V infection serves as a reminder that genotype V infection may contribute to hemorrhage in dengue patients.The relationship between DENV-1 genotype V infection and PLT loss was further estimated.The chemokines RANTES and GRO-α were released by activated PLT. 36Moreover, thrombocytopenia is a typical common symptom of SD. 37 Furthermore, a lower level of RANTES was observed in patients with SD and mice suffering from severe DENV infection. 38,39The loss of PLT was found to be closely correlated with a lower concentration of RANTES.In the study, lower levels of RANTES and GRO-α were observed in patients infected with DENV-1 genotype V, who were more prone to SD and bleeding.Above all, infection of DENV-1 genotype V could lead to a decrease in PLT, resulting in lower levels of RANTES and GRO-α.DENV-1 Genotype V might be relation to SD, especially SH.
As is well known, the inflammatory response plays a crucial role in the progression of SD. 40,41 Previous studies have suggested that inflammatory cytokines such as TNF-α, IFN-γ, and IL-10 are reported to be associated with the disease severity of dengue. 42,43Otherwise, sVCAM-1 is released from the endothelial cell surface into the circulation upon endothelial activation.It has now been proven to be a marker for endothelial injury during inflammatory processes. 44gher levels of cytokines and adhesion molecules mentioned above were detected in patients infected with genotype V during the early phase of illness.It provided evidence that Genotype V promotes the severe plasma leakage leading to shock or fluid accumulation with respiratory distress; (2) severe hemorrhage (SH); (3) severe organ impairment (SOI) as aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels ≥ 1000 U/L, impaired consciousness, heart and other organs impairment.The dengue illness is characterized by three phases: (1) febrile phase is between the 1st and 3rd day of onset; (2) critical phase is during the 4th to 6th day of onset; (3) recovery phase is after the 7th day of onset. 18The healthy individuals included three females and five males.All the healthy individuals were adults and the median age was 42 years old.The research protocol was approved by the ethics committee of the Guangzhou Eighth People's Hospital, Guangzhou Medical University (Reference Number 20100835, 20131224, and 20159264) and all the participants have written the informed consent forms.This study was retrospectively registered in the Chinese Clinical Trial Registry (ChiCTR2100046696).

T A B L E 1
Clinical characteristics of patients infected with different genotypes of DENV-1.

F I G U R E 3
Difference of Inflammatory mediator factors among the health individuals (NC), severe dengue patients infected with genotype I and severe dengue patients infected with genotype V. (A) tumor necrosis factor-α (TNF-α); (B) interferon-γ (IFN-γ); (C) interleukin-10 (IL-10); (D) growth related oncogene-α (GRO-α); (E) regulated upon activation normal T-cell expressed and secreted (RANTES); (F) soluble vascular cell adhesion molecule-1 (sVCAM-1).*p < 0.5, **p < 0.01, ***p < 0.001.secretion of inflammatory mediators, including TNF-α, IFN-γ, IL-10, and sVCAM-1.SD due to Genotype V infection may correlate with an excessive inflammatory response.Our study has several limitations.First, due to the retrospective study design, not all patients underwent measurements of inflammatory mediator factors and DENV viral load.Therefore, their role in potentially underestimating the immune response to DENV-1 genotype might be overlooked.Furthermore, the trend of inflammatory mediator factors during illness was still unknown.Second, according to the Dengue Guidance of 2009, the peak of viremia occurs on day 2 of illness and decreases rapidly.The delayed timing of serum collection in this study has significantly reduced the successful isolation of DENVs.Third, limited genomic sequencing of DENVs was conducted in the past due to poor conditions.It would be helpful for understanding the clinical manifestations of different genotypes of DENV-1.Last and but not least, the investigation of clinical fluid accumulation is limited by the lack of complementary radiographic data.To our best knowledge, this study is the first study to investigate the relationship between the DENV-1 genotype and disease severity.We have found that patients infected with DENV-1 genotype V showed elevated levels of inflammatory mediator factors, including TNF-α, IFN-γ, IL-10, and sVCAM-1, and decreased chemokines as RANTES and GRO-α.Furthermore, patients infected with DENV-1 genotype V were more frequently linked to SD, characterized by significant SOI.Our study, which elucidates the pathogenesis of SD through virology, could be valuable for the early admission of dengue patients and the development of anti-DENV drugs.Furthermore, the mechanism of the immune system response to DENV-1 genotypes would be assessed by vitro.and vivo. in the future.AUTHOR CONTRIBUTIONSXing-yu Leng (clinical cases collation, data analysis, writing); Ling-zhai Zhao (DENVs isolation, DENV genomic sequences performed); Lu Liao (clinical cases collation, data analysis, validation and verification); Kang-hong Jin (data validation and verification, manuscript proofread); Jia-min Feng (inflammatory mediator factors detection); Fu-chun Zhang contributed to the study concepts, design, manuscript preparation, manuscript editing, manuscript review.All authors have read and approved the final manuscript.ACKNOWLEDGMENTS This study was supported by Special Program of Science and Technology of Guangdong Province (Grant No.: 2013A020229001), F I G U R E 4 Difference of Inflammatory mediator factors among the health individuals (NC), severe dengue patients infected with genotype I and severe dengue patients infected with genotype V. (A) interleukin-15 (IL-15); (B) interleukin-17A (IL-17A); (C) interleukin-8 (IL-8); (D) monocyte chemotactic protein-1 (MCP-1).***p < 0.001.