The Relationship Between HTNV RNA Load In Patients Having Hemorrhagic Fever With Renal Syndrome And Severity Of The Disease

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.