肾综合征出血热患者血浆病毒载量的检测及其与病程病情关系的研究
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摘要
汉滩病毒是汉坦病毒属中的一个成员，感染其天然宿主条纹姬鼠后引起慢性无症状感染。然而，汉滩病毒感染人类引起以发热、出血、低血压和肾衰竭为典型临床特征的急性传染病-肾综合征出血热。全世界每年肾综合征出血热发病人数达10万左右，其中70～90%病例发生在中国，死亡率达0.1%-15%。肾综合征出血热疾病过程中有五个典型临床分期：发热期、休克期、少尿期、多尿期和恢复期。
肾综合征出血热的基本病理改变为全身小血管和毛细血管广泛损害、通透性增加，然而其致病机制至今仍未阐明。汉滩病毒引起的肾综合征出血热是一个多因素参与的过程，汉坦病毒感染宿主细胞导致的直接损伤以及病毒感染后引起的免疫病理损伤可能在全身小血管和毛细血管广泛损害中共同发挥作用，病毒感染直接作用包括改变内皮细胞的运动能力和通透性以及病毒影响机体的固有免疫应答，病毒感染引起机体的免疫应答包括免疫复合物沉积、补体激活、体液和T细胞免疫应答以及病毒感染诱导细胞释放炎症因子等。
除汉滩病毒外，其他病毒如汉城病毒、多普拉病毒、阿穆尔河病毒以及普马拉病毒感染人类也能引起肾综合征出血热。最近有一些研究表明汉坦病毒属中的多普拉病毒、普马拉病毒RNA载量与病情严重程度有相关性，然而至今没有汉滩病毒检测的商品化试剂盒，也未见有汉滩病毒引起的肾综合征出血热病毒载量与疾病严重程度及病理损伤关系的研究。
血浆细胞外游离DNA（cf-DNA）是存在于血浆中的细胞外双链DNA，主要来源于凋亡和坏死细胞，反应了机体受损程度，在多种急性和慢性疾病中升高并与疾病严重程度相关。最近研究表明，血浆cf-DNA在登革热以及普马拉病毒引起的肾综合征出血热等疾病中升高且与疾病严重程度相关。然而至今未见有汉滩病毒引起的肾综合征出血热患者血浆cf-DNA的动态变化规律及其与疾病严重程度相关性的研究。
本研究通过一步法实时定量RT-PCR方法检测2009～2011年第四军医大学唐都医院传染科101例患者的198份血浆标本的病毒载量，通过荧光染色法检测患者血浆标本的cf-DNA水平，通过捕获法ELISA检测患者血清中抗汉滩病毒核蛋白的IgM和IgG的效价，并分别分析了患者血浆病毒载量和血浆cf-DNA与患者血小板计数、肌酐、白细胞计数之间的相关性，患者血浆病毒载量和血浆cf-DNA之间的相关性，以及血浆病毒载量和血清IgM和IgG的效价之间的相关性。
（1）建立了检测汉滩病毒载量的一步法实时定量RT-PCR方法，检测灵敏度为2700copies/mL血浆，该方法特异性良好，汉城病毒L99株和常见病毒HBV、HCV、流感病毒不会引起假阳性，批内差异和批间差异分别为：0.6%～4.5%和3.9%～7.3%，重复性好。
（2）检测了2009～2011年唐都医院住院101例HFRS患者（男78例，女23例，年龄跨度9～73岁）198份血浆标本的病毒载量和其中有两个病期以上患者170份血浆标本的cf-DNA水平：①提取患者血浆标本RNA后用建立的一步法实时定量RT-PCR检测病毒载量，79例患者的84份标本能检测到病毒，病毒载量为3.43～7.33log10copies/mL。②患者血浆直接荧光染色法检测均能检测到cf-DNA的存在，浓度范围为：832～41400ng/mL。
（3）依据临床症状和实验室检测指标将患者分为轻型/中型和重型/危重型两组，分别分析了肾综合征出血热患者血浆病毒载量和血浆cf-DNA与病程、病情和病情指标之间的相关性，患者血浆病毒载量和血浆cf-DNA之间的相关性，以及血浆病毒载量和血清IgM和IgG的效价之间的相关性，发现：①汉滩病毒载量在发热期、低血压休克期明显升高，随病程进展到少尿期迅速下降，多尿期与恢复期检测不到病毒载量，这与汉滩病毒感染的重要特征-自限性一致。②疾病早期重型/危重型患者血浆病毒载量高于轻型/中型患者(分别为5.90和5.03log10copies/mL; P=0.001)，患者发热期/休克期病毒载量与血小板计数最低值呈负相关，与肌酐最高值呈正相关，提示汉滩病毒感染病毒载量与疾病严重程度相关。③用GST捕获法检测患者血清IgM、IgG的效价，并分析了患者发热期/休克期病毒载量与患者血清特异性IgM和IgG效价的峰值的相关性，没有发现明显相关性。④患者血浆cf-DNA疾病早期升高，病程进展至多尿期cf-DNA下降至正常水平。⑤疾病早期重型/危重型患者血浆cf-DNA高于轻型/中型患者（3664vs.2060ng/mL，P=0.007），患者发热期/休克期血浆cf-DNA与白细胞计数最高值呈正相关，与血小板计数最低值呈负相关，与肌酐最高值呈正相关，提示汉滩病毒感染后血浆cf-DNA水平与疾病严重程度相关。⑥患者血浆cf-DNA与血浆病毒载量明显正相关(P<0.001； r=0.68)，提示汉滩病毒感染后引起的细胞的坏死或凋亡可能参与疾病的病理损伤。
本研究首次系统检测汉滩病毒引起的肾综合征出血热患者血浆病毒载量和血浆cf-DNA水平，并分别分析了病毒载量和血浆cf-DNA与病程、病情之间的相关性，患者血浆病毒载量和血浆cf-DNA之间的相关性，以及血浆病毒载量和血清IgM和IgG的效价之间的相关性，结果表明：汉滩病毒引起的肾综合征出血热患者发热期/休克期血浆病毒载量和血浆cf-DNA与疾病严重程度相关，可以作为预测疾病预后的指标，明确了汉滩病毒载量和血浆cf-DNA在肾综合征出血热中的变化规律，并为研究汉滩病毒在肾综合征出血热致病机制中的作用提供理论和实验依据。
Hantaan virus (HTNV), a member of the genus Hantavirus, causes a chronic,asymptomatic infection in its natural host, the striped field mouse Apodemusagrarius. In contrast, HTNV infection in humans manifests as acutehemorrhagic fever with renal syndrome (HFRS). Typically, HFRS occurs in fivesequential stages: febrile, hypotensive, oliguric, diuretic, and convalescent.Clinical symptoms also include thrombocytopenia and, in severe cases,hemorrhage caused by capillary leak syndrome. As many as100,000cases ofHFRS have occurred annually worldwide, of which over70%～90%weredocumented in the mainland of China, with a mortality rate of0.1%-15%.
The pathogenesis of HFRS is considerably far from being completelyunderstood. The basic mechanisms underlying HFRS pathogenesis relate toincreased vascular permeability, and it has been widely recognized recently thatviral replication together with the immune response, which involving immunecomplexes, complement activation, B cell response, T cell response, and HTNV-induced cytokine production, are involved in tissue injury.
HFRS could be caused by other Hantaviruses besides HTNV, such asDobrava virus, Seoul virus, Amur virus, and Puumala virus. Some recent studieshave suggested that there might be an association between the Hantavirus RNAload and severity of the corresponding disease. Nevertheless, there has been noreport until now about the quantitative HTNV RNA load in HFRS patients,including the relationship between HTNV load and the pathogenesis andseverity of HFRS.
Cell free DNA (cf-DNA) is a kind of extracellular double strain DNAexisting in the plasma. Cf-DNA is released by the apoptosis or necrosis cells,increased in several acute or chronic diseases, and related with the diseaseseverity. Recent studies demonstrated that plasma cf-DNA is increased inDengue Fever and HFRS caused by Puumala virus, and is positively related withthe disease severity. However, there hasn’t been any report concerning thedynamic changes of cf-DNA or its relationship with disease severity in HFRScaused by HTNV.
In our present study, the HTNV RNA load was measured in plasmasamples from HFRS patients with a quantitative one-step real-timereverse-transcriptase polymerase chain reaction (RT-PCR) assay. The cf-DNAlevel in patients’ plasma was detected by fluorescent staining, and therelationship between viral load or cf-DNA and disease course and diseaseseverity (including clinical symptom as well as laboratory parameters such ascreatinine, platelet count, and white cells count), the relationship between viralload and cf-DNA, and the relationship between viral load and level of specifichumoral immunity were analyzed respectively. The details are as following:
1. One-step real-time quantitative RT-PCR assay was established and used to detect the HTNV RNA load. The detect limit of the assay reached2700copies/mL (3.43log10copies/mL) in the plasma and the assay showed nocross-reactivity to4other viruses, namely, HBV, HCV, influenza, and Seoulviruses. Furthermore, the intra-assay and inter-assay variabilities of the assaywere evaluated to be ranging from0.6%to4.5%and3.9%to7.3%, respectively,which indicated the high reproducibility of the assay.
2. A panel of198plasma samples were collected from101patients (78male and23female subjects in the age range of9-73years) with HFRSaperiodically during hospitalization. Virus RNA was extracted from the plasmasamples of HFRS patients and the HTNV RNA load was detected by theRT-PCR assay mentioned above. Among the198plasma samples from101patients which were examined,84samples from79patients could be detectedfor HTNV RNA load, ranging from3.43to7.33log10copies per mL of plasma.Meanwhile, cf-DNA level in plasma from patients who have samples in overtwo disease stages (altogether170samples) was detected, and the cf-DNA in allthe170plasma samples could be detected by direct fluorescent staining, rangingfrom832to41400ng/ml.
3. The patients were categorized as having severe-to-critical disease (i.e.,the severe/critical group) or mild-to-moderate disease (i.e., the mild/moderategroup), on the basis of clinical and laboratory parameters. The relationshipbetween viral load or cf-DNA and disease course and disease severity (includingclinical symptom as well as laboratory parameters), the relationship betweenviral load and cf-DNA, and the relationship between viral load and level ofspecific humoral immunity were analyzed.①HTNV RNA load could only bedetected in plasma of HFRS patients in febrile/hypotensive and oliguric stageand it gradually declined and decreased to an undetectable level with the progress of the disease, which is in accordance with the most important propertyof HFRS, that is, self-limiting.②In the early stage of disease, patients insevere/critical group were found to have higher viral loads than those inmild/moderate group (5.90vs.5.03log10copies/mL; P=0.001), suggesting anassociation between Hantaan virus load and disease severity. And patients withhigh viral loads in febrile/hypotensive stage might be more likely to have a moresevere course of disease. We also found that viral load in the febrile/hypotensivestage was positively related with the peak value of serum creatinine andnegatively related with the lowest value of platelet count, which further certifiedthe relationship between viral loads and disease severity.③The relationshipbetween HTNV viral load in febrile/hypotensive samples and the peak value oftiters of HTNV-NP specific IgM and IgG was also investigated using a GSTcapture ELISA method. No significant correlation between antibody titers andviral load was found.④cf-DNA level in plasma from HFRS patients wasincreased in early stages of the disease and gradually decreased to normal levelin diuretic stage.⑤In the early stage of disease, patients in severe/critical groupwere found to have higher cf-DNA level than those in mild/moderate group(3664vs.2060ng/mL; P=0.007), and cf-DNA level in the febrile/hypotensivestage was positively related with the peak value of serum creatinine and thewhile cells count, and negatively related with the lowest value of platelet count,suggesting an association between cf-DNA level and disease severity afterHTNV infection.⑥A significant positive relationship between cf-DNA andviral load in plasma has also been found (P<0.001; r=0.68), indicating thatcellular apoptosis or necrosis induced after HTNV infection might be involvedin the pathogenesis of HFRS.
In conclusion, this is the first study to describe the plasma HTNV RNA load and cf-DNA level in HFRS patients and discuss their relationship between eachother and with disease course, disease severity, and level of specific humoralimmunity based on a kinetic observation. It is indicated that the viral load andcf-DNA in plasma from HFRS patients are both related with the disease severityand could be used as a prognostic marker for monitoring and/or predicting theseverity of clinical manifestations. These results provide experimental data forfully understanding the role of HTNV RNA load as well as plasma cf-DNA inHFRS, and establish the foundation for further exploration of the relationshipbetween HTNV load and the pathogenesis and severity of HFRS.
肾综合征出血热的基本病理改变为全身小血管和毛细血管广泛损害、通透性增加，然而其致病机制至今仍未阐明。汉滩病毒引起的肾综合征出血热是一个多因素参与的过程，汉坦病毒感染宿主细胞导致的直接损伤以及病毒感染后引起的免疫病理损伤可能在全身小血管和毛细血管广泛损害中共同发挥作用，病毒感染直接作用包括改变内皮细胞的运动能力和通透性以及病毒影响机体的固有免疫应答，病毒感染引起机体的免疫应答包括免疫复合物沉积、补体激活、体液和T细胞免疫应答以及病毒感染诱导细胞释放炎症因子等。
除汉滩病毒外，其他病毒如汉城病毒、多普拉病毒、阿穆尔河病毒以及普马拉病毒感染人类也能引起肾综合征出血热。最近有一些研究表明汉坦病毒属中的多普拉病毒、普马拉病毒RNA载量与病情严重程度有相关性，然而至今没有汉滩病毒检测的商品化试剂盒，也未见有汉滩病毒引起的肾综合征出血热病毒载量与疾病严重程度及病理损伤关系的研究。
血浆细胞外游离DNA（cf-DNA）是存在于血浆中的细胞外双链DNA，主要来源于凋亡和坏死细胞，反应了机体受损程度，在多种急性和慢性疾病中升高并与疾病严重程度相关。最近研究表明，血浆cf-DNA在登革热以及普马拉病毒引起的肾综合征出血热等疾病中升高且与疾病严重程度相关。然而至今未见有汉滩病毒引起的肾综合征出血热患者血浆cf-DNA的动态变化规律及其与疾病严重程度相关性的研究。
本研究通过一步法实时定量RT-PCR方法检测2009～2011年第四军医大学唐都医院传染科101例患者的198份血浆标本的病毒载量，通过荧光染色法检测患者血浆标本的cf-DNA水平，通过捕获法ELISA检测患者血清中抗汉滩病毒核蛋白的IgM和IgG的效价，并分别分析了患者血浆病毒载量和血浆cf-DNA与患者血小板计数、肌酐、白细胞计数之间的相关性，患者血浆病毒载量和血浆cf-DNA之间的相关性，以及血浆病毒载量和血清IgM和IgG的效价之间的相关性。
（1）建立了检测汉滩病毒载量的一步法实时定量RT-PCR方法，检测灵敏度为2700copies/mL血浆，该方法特异性良好，汉城病毒L99株和常见病毒HBV、HCV、流感病毒不会引起假阳性，批内差异和批间差异分别为：0.6%～4.5%和3.9%～7.3%，重复性好。
（2）检测了2009～2011年唐都医院住院101例HFRS患者（男78例，女23例，年龄跨度9～73岁）198份血浆标本的病毒载量和其中有两个病期以上患者170份血浆标本的cf-DNA水平：①提取患者血浆标本RNA后用建立的一步法实时定量RT-PCR检测病毒载量，79例患者的84份标本能检测到病毒，病毒载量为3.43～7.33log10copies/mL。②患者血浆直接荧光染色法检测均能检测到cf-DNA的存在，浓度范围为：832～41400ng/mL。
（3）依据临床症状和实验室检测指标将患者分为轻型/中型和重型/危重型两组，分别分析了肾综合征出血热患者血浆病毒载量和血浆cf-DNA与病程、病情和病情指标之间的相关性，患者血浆病毒载量和血浆cf-DNA之间的相关性，以及血浆病毒载量和血清IgM和IgG的效价之间的相关性，发现：①汉滩病毒载量在发热期、低血压休克期明显升高，随病程进展到少尿期迅速下降，多尿期与恢复期检测不到病毒载量，这与汉滩病毒感染的重要特征-自限性一致。②疾病早期重型/危重型患者血浆病毒载量高于轻型/中型患者(分别为5.90和5.03log10copies/mL; P=0.001)，患者发热期/休克期病毒载量与血小板计数最低值呈负相关，与肌酐最高值呈正相关，提示汉滩病毒感染病毒载量与疾病严重程度相关。③用GST捕获法检测患者血清IgM、IgG的效价，并分析了患者发热期/休克期病毒载量与患者血清特异性IgM和IgG效价的峰值的相关性，没有发现明显相关性。④患者血浆cf-DNA疾病早期升高，病程进展至多尿期cf-DNA下降至正常水平。⑤疾病早期重型/危重型患者血浆cf-DNA高于轻型/中型患者（3664vs.2060ng/mL，P=0.007），患者发热期/休克期血浆cf-DNA与白细胞计数最高值呈正相关，与血小板计数最低值呈负相关，与肌酐最高值呈正相关，提示汉滩病毒感染后血浆cf-DNA水平与疾病严重程度相关。⑥患者血浆cf-DNA与血浆病毒载量明显正相关(P<0.001； r=0.68)，提示汉滩病毒感染后引起的细胞的坏死或凋亡可能参与疾病的病理损伤。
本研究首次系统检测汉滩病毒引起的肾综合征出血热患者血浆病毒载量和血浆cf-DNA水平，并分别分析了病毒载量和血浆cf-DNA与病程、病情之间的相关性，患者血浆病毒载量和血浆cf-DNA之间的相关性，以及血浆病毒载量和血清IgM和IgG的效价之间的相关性，结果表明：汉滩病毒引起的肾综合征出血热患者发热期/休克期血浆病毒载量和血浆cf-DNA与疾病严重程度相关，可以作为预测疾病预后的指标，明确了汉滩病毒载量和血浆cf-DNA在肾综合征出血热中的变化规律，并为研究汉滩病毒在肾综合征出血热致病机制中的作用提供理论和实验依据。
Hantaan virus (HTNV), a member of the genus Hantavirus, causes a chronic,asymptomatic infection in its natural host, the striped field mouse Apodemusagrarius. In contrast, HTNV infection in humans manifests as acutehemorrhagic fever with renal syndrome (HFRS). Typically, HFRS occurs in fivesequential stages: febrile, hypotensive, oliguric, diuretic, and convalescent.Clinical symptoms also include thrombocytopenia and, in severe cases,hemorrhage caused by capillary leak syndrome. As many as100,000cases ofHFRS have occurred annually worldwide, of which over70%～90%weredocumented in the mainland of China, with a mortality rate of0.1%-15%.
The pathogenesis of HFRS is considerably far from being completelyunderstood. The basic mechanisms underlying HFRS pathogenesis relate toincreased vascular permeability, and it has been widely recognized recently thatviral replication together with the immune response, which involving immunecomplexes, complement activation, B cell response, T cell response, and HTNV-induced cytokine production, are involved in tissue injury.
HFRS could be caused by other Hantaviruses besides HTNV, such asDobrava virus, Seoul virus, Amur virus, and Puumala virus. Some recent studieshave suggested that there might be an association between the Hantavirus RNAload and severity of the corresponding disease. Nevertheless, there has been noreport until now about the quantitative HTNV RNA load in HFRS patients,including the relationship between HTNV load and the pathogenesis andseverity of HFRS.
Cell free DNA (cf-DNA) is a kind of extracellular double strain DNAexisting in the plasma. Cf-DNA is released by the apoptosis or necrosis cells,increased in several acute or chronic diseases, and related with the diseaseseverity. Recent studies demonstrated that plasma cf-DNA is increased inDengue Fever and HFRS caused by Puumala virus, and is positively related withthe disease severity. However, there hasn’t been any report concerning thedynamic changes of cf-DNA or its relationship with disease severity in HFRScaused by HTNV.
In our present study, the HTNV RNA load was measured in plasmasamples from HFRS patients with a quantitative one-step real-timereverse-transcriptase polymerase chain reaction (RT-PCR) assay. The cf-DNAlevel in patients’ plasma was detected by fluorescent staining, and therelationship between viral load or cf-DNA and disease course and diseaseseverity (including clinical symptom as well as laboratory parameters such ascreatinine, platelet count, and white cells count), the relationship between viralload and cf-DNA, and the relationship between viral load and level of specifichumoral immunity were analyzed respectively. The details are as following:
1. One-step real-time quantitative RT-PCR assay was established and used to detect the HTNV RNA load. The detect limit of the assay reached2700copies/mL (3.43log10copies/mL) in the plasma and the assay showed nocross-reactivity to4other viruses, namely, HBV, HCV, influenza, and Seoulviruses. Furthermore, the intra-assay and inter-assay variabilities of the assaywere evaluated to be ranging from0.6%to4.5%and3.9%to7.3%, respectively,which indicated the high reproducibility of the assay.
2. A panel of198plasma samples were collected from101patients (78male and23female subjects in the age range of9-73years) with HFRSaperiodically during hospitalization. Virus RNA was extracted from the plasmasamples of HFRS patients and the HTNV RNA load was detected by theRT-PCR assay mentioned above. Among the198plasma samples from101patients which were examined,84samples from79patients could be detectedfor HTNV RNA load, ranging from3.43to7.33log10copies per mL of plasma.Meanwhile, cf-DNA level in plasma from patients who have samples in overtwo disease stages (altogether170samples) was detected, and the cf-DNA in allthe170plasma samples could be detected by direct fluorescent staining, rangingfrom832to41400ng/ml.
3. The patients were categorized as having severe-to-critical disease (i.e.,the severe/critical group) or mild-to-moderate disease (i.e., the mild/moderategroup), on the basis of clinical and laboratory parameters. The relationshipbetween viral load or cf-DNA and disease course and disease severity (includingclinical symptom as well as laboratory parameters), the relationship betweenviral load and cf-DNA, and the relationship between viral load and level ofspecific humoral immunity were analyzed.①HTNV RNA load could only bedetected in plasma of HFRS patients in febrile/hypotensive and oliguric stageand it gradually declined and decreased to an undetectable level with the progress of the disease, which is in accordance with the most important propertyof HFRS, that is, self-limiting.②In the early stage of disease, patients insevere/critical group were found to have higher viral loads than those inmild/moderate group (5.90vs.5.03log10copies/mL; P=0.001), suggesting anassociation between Hantaan virus load and disease severity. And patients withhigh viral loads in febrile/hypotensive stage might be more likely to have a moresevere course of disease. We also found that viral load in the febrile/hypotensivestage was positively related with the peak value of serum creatinine andnegatively related with the lowest value of platelet count, which further certifiedthe relationship between viral loads and disease severity.③The relationshipbetween HTNV viral load in febrile/hypotensive samples and the peak value oftiters of HTNV-NP specific IgM and IgG was also investigated using a GSTcapture ELISA method. No significant correlation between antibody titers andviral load was found.④cf-DNA level in plasma from HFRS patients wasincreased in early stages of the disease and gradually decreased to normal levelin diuretic stage.⑤In the early stage of disease, patients in severe/critical groupwere found to have higher cf-DNA level than those in mild/moderate group(3664vs.2060ng/mL; P=0.007), and cf-DNA level in the febrile/hypotensivestage was positively related with the peak value of serum creatinine and thewhile cells count, and negatively related with the lowest value of platelet count,suggesting an association between cf-DNA level and disease severity afterHTNV infection.⑥A significant positive relationship between cf-DNA andviral load in plasma has also been found (P<0.001; r=0.68), indicating thatcellular apoptosis or necrosis induced after HTNV infection might be involvedin the pathogenesis of HFRS.
In conclusion, this is the first study to describe the plasma HTNV RNA load and cf-DNA level in HFRS patients and discuss their relationship between eachother and with disease course, disease severity, and level of specific humoralimmunity based on a kinetic observation. It is indicated that the viral load andcf-DNA in plasma from HFRS patients are both related with the disease severityand could be used as a prognostic marker for monitoring and/or predicting theseverity of clinical manifestations. These results provide experimental data forfully understanding the role of HTNV RNA load as well as plasma cf-DNA inHFRS, and establish the foundation for further exploration of the relationshipbetween HTNV load and the pathogenesis and severity of HFRS.
引文
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