【前言】登革病毒属于黄病毒科黄病毒属，是一种RNA病毒，感染人体后主要引起登革热以及发病率和死亡率都很高的登革出血热和登革休克综合征。登革热已成为全球性公共卫生挑战，据世界卫生组织统计，全球逾100个国家和地区曾报告过登革热疫情爆发。自2008年起，云南省持续报告登革热疫情，呈现多年连续流行的态势。
【目的】2023年，云南省西双版纳州爆发较大规模登革热疫情，累计报告确诊病例6628例。本研究针对当地流行株的分子特征进行分析，以探究病毒进化与疫情爆发之间的关联。此外，基于课题组前期筛选获得的2型登革病毒冷适应减毒株中发现的突变位点，我们针对其中位于prM蛋白中的两个位点，构建并拯救了双突变克隆株，并在细胞及动物水平对突变株进行毒力评价，尝试探索DENV毒力减弱的机制和可能性。
【方法】本研究采集2023年西双版纳州人民医院经过NS1试剂盒检测阳性、疑似登革热阳性的患者血清，使用QIAmp 病毒RNA提取试剂盒提取病毒RNA。首先用探针qPCR的方法进行登革病毒4种血清型的分型鉴定，同时对登革病毒、寨卡病毒、乙型脑炎病毒、基孔肯雅病毒共4种黄病毒的共感染情况进行鉴别。此后，使用TAKARA的逆转录试剂盒将病毒RNA逆转录成cDNA，针对全长设计18对引物进行分段PCR扩增，将所得条带送生工生物公司进行Sanger测序法测序，将所得序列进行拼接，得到登革病毒的全长基因组序列。对序列进行碱基组成和氨基酸组成的分析，在NCBI数据库中通过BLAST选择相似度较高的序列，使用MEGA11软件构建进化树。结合我国国内各年流行株分析各蛋白上存在的突变，使用网站工具预测二级结构和B细胞线性表位，计算蛋白编码序列的选择压力，关注结构蛋白和非结构蛋白分别发生的进化事件。此外，本研究使用点突变试剂盒克隆构建出2型登革病毒双突变克隆株DENV2-5M，使用单酶切的方法线性化，使用共转录加帽试剂盒进行体外转录，最终在Vero细胞中进行拯救。获得DENV2-5M毒株后，在Vero细胞中测定其增殖曲线和感染性滴度的变化，与原毒株DENV2-37进行对比，验证是否毒力减弱。建立动物模型，同时使用突变株DENV2-5M和原毒株DENV2-37感染5周龄的A129小鼠， 对小鼠的体重变化和临床评分进行记录，在第5天处死，取重要组织器官和全血进行病毒载量的测定；在第7天处死第二批，取器官和全血进行病毒载量的测定，同时取重要组织器官进行组织病理学的检测。此外还针对DENV2-5M和DENV2-37进行了中和抗体的检测。
【结果】引起2023年西双版纳登革疫情的主要流行株是1型登革病毒，本实验共分离得到了23CNYN1-10共10株病毒的全基因组序列，其中有23CNYN2、23CNYN5-10这7条序列完全相同，23CNYN1-4之间的碱基突变位点分布在prM、E、NS2A、NS2B、NS3、NS4A、NS5的编码序列中，氨基酸突变位点分布在prM、NS2A、NS3、NS4A、NS5蛋白上。在预测结果中，与标准株DQ672562相比，23CNYN1、2的prM二级结构发生改变，蛋白质结合位点增加；NS1的二级结构没有发生明显改变，RNA结合位点数量保持不变。B细胞线性表位预测的结果显示标准株DQ672562与23CNYN1之间也存在差异，有表位数量和评分的改变。在第二部分，构建出双突变的克隆株DENV2-5M，并成功在Vero细胞上进行了拯救。利用Vero细胞分别绘制DENV2-5M和DENV2-37的增殖动力学曲线，发现从DENV2-5M表现出增殖速度较DENV2-37更慢的特点，在观察的7天中DENV2-5M的增殖曲线始终低于DENV2-37。同时每天收取一组上清样品用于进行感染性滴度的计算，DENV2-37峰值较高，到达较快，DENV2-5M峰值较低，但相差不多，到达峰值要比DENV2-37晚一天。此后使用皮下多点注射的方式建立动物模型，在动物模型中进行了体重变化、组织病毒载量、组织病理学的检测。结果显示DENV2-37对A129小鼠的体重变化影响较大，在注射早期会使小鼠的体重下降，DENV2-5M对A129小鼠的体重变化影响不大，没有使小鼠体重有明显下降，使小鼠体重在一个范围内稳定。在组织病毒载量的比较中，选择了感染后5天和7天的脾、肝、肺、小肠和全血提取RNA进行RT-qPCR的实验，在各组中都显示DENV2-37的病毒载量高于DENV2-5M的病毒载量。其中在脾和肝中，第7天的病毒载量差远高于第5天的病毒载量差，在第7天时DENV2-37组的病毒载量达到DENV2-5M组的病毒载量的10倍以上，在第5天时只有3倍左右；在全血中第5天和第7天的病毒载量差相差不多DENV2-37组均达到DENV2-5M组的9.5倍左右，在肺中的病毒载量第5天DENV2-37组达到DENV2-5M组的2倍，第7天达到4倍；在小肠中的病毒载量第5天DENV2-37组达到DENV2-5M组的6倍，第7天达到3倍。在肺中通过组织病理学的观察，发现在肺组织中DENV2-37组呈现较DENV2-5M组更明显的肺泡壁增厚；在脾组织中DENV2-5M较MOCK组表现出少量的粒细胞浸润，DENV2-37组较DENV2-5M组表现出更明显的粒细胞浸润；在肝组织中DENV2-5M组有细胞肿胀、胞核固缩的病变，DENV2-37组还存在有细胞坏死和更多的粒细胞浸润；在肾组织中DENV2-5M组与DENV2-37组的病变均不明显；在肠组织中DENV2-5M与DENV2-37组均表现出肠绒毛与固有层分离，DENV2-37组有更多的粒细胞浸润。
【结论】进化树分析显示2023年云南西双版纳1型登革病毒流行株与同年在广东流行的PP563875毒株最为接近，距离最近的国外流行株是2021年在泰国流行的MZ619041毒株，推测2023年我国云南省西双版纳州的登革病毒可能起源于东南亚。冷适应减毒株上发现的位于prM福林酶切位点附近的两个突变可以造成2型登革病毒的毒力减弱。

Background: Dengue virus belongs to the genus flavivirus of flaviviridae, which is an RNA virus. It mainly causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome with high incidence rate and mortality after infection. Currently, over 100 countries have reported the prevalence of dengue fever, which has become a global public health issue. Since 2008, there have been continuous outbreaks of dengue fever in Yunnan.
Objective: In 2023, Xishuangbanna Prefecture, Yunnan Province, experienced a DENV-1-associated dengue outbreak with 6,628 laboratory-confirmed cases. This study systematically investigates the genomic characterization of epidemic DENV-1 strains circulating in the region through comprehensive phylogenetic and molecular clock analyses, aiming to elucidate the evolutionary dynamics driving virus propagation and epidemic intensity. In addition, based on the mutation sites found in the cold adapted attenuated strain of type 2 dengue virus, we constructed and rescued a double mutant clone targeting two sites in the prM protein, and evaluated the virulence of the mutant strain at the cellular and animal levels, attempting to explore the mechanism and possibility of DENV virulence reduction.
Method: This study collected serum samples from patients who tested positive for NS1 and suspected to be positive for dengue fever at Xishuangbanna People's Hospital in 2023. The QIAmp virus RNA extraction kit was used to extract viral RNA. Firstly, qPCR was used to classify and identify the four serotypes of dengue virus, and co infection identification of four yellow viruses including dengue virus, Zika virus, Japanese encephalitis virus, and chikungunya virus was also performed. Afterwards, the virus RNA was reverse transcribed into cDNA using TAKARA's reverse transcription kit, and 18 pairs of primers were designed for segmented PCR amplification of the full-length. The resulting bands were sent to a biotech company for Sanger sequencing, and the sequences were concatenated to obtain the full-length genome sequence of the virus. Analyze the base and amino acid composition of the sequence, select sequences with high similarity using BLAST in the NCBI database, and construct an evolutionary tree using MEGA11 software. Based on the analysis of prevalent strains in various years in China, mutations in various proteins were identified. Website tools were used to predict secondary structures and B cell linear epitopes, calculate the selection pressure of protein coding sequences, and focus on the evolutionary events that occur in structural and non structural proteins, respectively. In addition, this study used a point mutation kit to clone and construct a type 2 dengue virus double mutant clone strain DENV2-5M. The strain was linearized using a single enzyme digestion method and transcribed in vitro using a co transcription cap kit. Finally, it was rescued in Vero cells. After obtaining the DENV2-5M strain, the changes in its proliferation curve and infectivity titer were measured in Vero cells, and compared with the original strain DENV2-37 to verify whether the virulence was weakened. Establish an animal model and infect 5-week-old A129 mice with mutant strain DENV2-5M and original strain DENV2-37 simultaneously. Record the weight changes and clinical scores of the mice, euthanize them on the 5th day, and measure the viral load in organs and whole blood; On the 7th day, the second batch was euthanized, and organs and whole blood were collected for virus load measurement. At the same time, organs were collected for histopathological examination. In addition, neutralizing antibodies were tested for DENV2-5M and DENV2-37.
Result: The main pathogenic strain of dengue fever in Xishuangbanna in 2023 is type 1 dengue virus. In this experiment, the whole genome sequences of 10 strains of virus, including 23CNYN1-10, were isolated. Among them, 7 sequences, 23CNYN2 and 23CNYN5-10, were completely identical. The base mutation sites between 23CNYN1-4 were distributed in the coding sequences of prM, E, NS2A, NS2B, NS3, NS4A, and NS5, and the amino acid mutation sites were distributed in the proteins of prM, NS2A, NS3, NS4A, and NS5. In the predicted results, compared with the standard strain DQ672562, the secondary structure of 23CNYN1 and 2prM changed, and the protein binding sites increased; The secondary structure of NS1 did not undergo significant changes, and the number of RNA binding sites remained unchanged. The results of linear epitope prediction for B cells also showed differences between the standard strain DQ672562 and 23CNYN1, with changes in the number of epitopes and scores. In the second part, a double mutant clone strain DENV2-5M was constructed and successfully rescued on Vero cells. Using Vero cells, the proliferation kinetics curves of DENV2-5M and DENV2-37 were plotted separately. It was found that DENV2-5M exhibited a slower proliferation rate than DENV2-37, and the proliferation curve of DENV2-5M was consistently lower than that of DENV2-37 during the 7-day observation period. At the same time, a set of supernatant samples is collected daily for the calculation of infectious titers. DENV2-37 has a higher peak and reaches it quickly, while DENV2-5M has a lower peak but is similar, reaching its peak one day later than DENV2-37. Subsequently, an animal model was established using subcutaneous multi-point injection, and weight changes, tissue viral load, and histopathological examination were performed in the animal model. The results showed that DENV2-37 had a significant impact on the weight changes of A129 mice, causing weight loss in the early stages of injection. DENV2-5M had little effect on the weight changes of A129 mice and did not cause significant weight loss, stabilizing the weight of mice within a certain range. In the comparison of viral load in tissues, RNA was extracted from spleen, liver, lung, small intestine, and whole blood at 5 and 7 days after infection for RT qPCR experiments. The viral load of DENV2-37 was higher than that of DENV2-5M in all groups. In the spleen and liver, the difference in viral load on the 7th day was much higher than that on the 5th day. On the 7th day, the viral load of the DENV2-37 group reached more than 10 times that of the DENV2-5M group, while on the 5th day it was only about 3 times; The difference in viral load between the 5th and 7th day in whole blood was similar, with the DENV2-37 group reaching about 9.5 times that of the DENV2-5M group. The viral load in the lungs was twice that of the DENV2-5M group on the 5th day and four times that on the 7th day; The viral load in the small intestine reached 6 times that of the DENV2-5M group on the 5th day and 3 times that of the DENV2-37 group on the 7th day. Through histopathological observation in the lungs, it was found that the DENV2-37 group exhibited more significant thickening of alveolar walls compared to the DENV2-5M group; In the spleen tissue, DENV2-5M showed a small amount of granulocyte infiltration compared to MOCK group, while DENV2-37 group showed more obvious granulocyte infiltration compared to DENV2-5M group; In the liver tissue, the DENV2-5M group showed cellular swelling and nuclear condensation, while the DENV2-37 group also exhibited cell necrosis and more granulocyte infiltration; The lesions in both DENV2-5M group and DENV2-37 group were not significant in renal tissue; Both DENV2-5M and DENV2-37 groups showed separation of intestinal villi and lamina propria in intestinal tissue, with more granulocyte infiltration in the DENV2-37 group.
Conclusion: The closest strain to the 2023 Yunnan Xishuangbanna type 1 dengue virus strain in the evolutionary tree is PP563875 (Guangdong, China), and the closest foreign strain is MZ619041 (Thailand, 2021). It is speculated that the dengue virus transmission in Xishuangbanna，Yunnan Province，China in 2023 may have originated from Southeast Asia. The mutation of the Fulin cleavage site on prM can cause a decrease in the virulence of type 2 dengue virus.