Discussion
Hemorrhagic fever with renal syndrome (HFRS), a global public health concern with a high fatality rate, has been reported in various countries. Approximately 90% of all the worldwide cases have been reported in China distributed in different regions, except Qinghai Province [5, 8, 12]. The highest incidences of HFRS in Shenyang, Anshan, Dandong, Jinzhou, Yingkou, and Huludao of Liaoning Province in China[13]. Meanwhile, another viral hemorrhagic fever infectious disease, SFTS, also affected Liaoning province. Therefore, exploring the relationship between co-infection of both epidemic viral haemorrhagic fever in severe clinical cases will contribute to differential diagnosis and treatment of the disease.
This study systematically analyzed the clinical and etiological characteristics of severe SFTS in Dandong City of Liaoning province. The results showed that the severe rate among HFRS patients co-infected with viral hemorrhagic fevers was higher than independent infection cases with no statistically significant difference. However, blood diagnostic detections revealed that 2 of the 3 severe HFRS patients who tested positive for HYNV were simultaneously positive for SFTSV. The results suggested that co-infection with both viral hemorrhagic fevers is associated with the occurrence of severe cases. Historically, there has been a consistent prevalence of vector-borne infectious diseases in Dandong, located in the Changbai Mountain region, which exhibits high forest coverage and encompasses abundant vectors such as rats and ticks. Previous studies indicated that the dominant tick variety in this region is Haemaphysalis longicornis, the carrier of the zoonotic pathogen SFTSV[11, 14]. This agent resides on the surfaces of rats’ bodies and transfers infectious diseases to humans through host activity. Moreover, ticks can spread disease rapidly and over long distances with migratory bird hosts, potentially leading to widespread disease outbreaks[15]. Various livestock, poultry, wild mammals, and rodents can naturally acquire SFTSV and present seropositivity under subclinical infection, revealing brief viremia and complete viral clearance after recovery[16,17]. This conflicts with our negative nucleic acid analysis of 167 captured wild mouse samples, indicating that wild mice are not directly involved in SFTSV transmission but may contract it from ticks feeding them.
Some previous studies have shown that the spatio-temporal distribution characteristics and clinical symptoms of the above two viral hemorrhagic fevers are comparable, leading to missed and erroneous clinical practice diagnoses. Inaccuracy of diagnosis affects effective disease treatment, hinders disease prognosis, and increases mortality risks. A retrospective analysis study conducted by Rui Qi et al. [18] revealed that SFRS patients were misdiagnosed as HRFS based on 73 (57.0%) having HTNV-IgM antibodies, and 4 (7.3%) were positive for both HTNV-IgM and SFTSV-IgM antibodies after evaluating 128 clinical HFRS patients.
In clinical practice, it is difficult to differentiate between HFRS and SFTS patients because of similar presentations. Patients with HFRS experience typical or atypical symptoms. Recently, atypical presentations predominate, showing mild symptoms similar to influenza, such as fever, fatigue, and headache, inviting potential misinterpretation [19, 20]. Moreover, HFRS and SFTS are both viral hemorrhagic fevers with similar mechanisms. The core of HFRS pathogenesis is endothelial cell infection by hantavirus, triggering a severe and rapid immune response resulting in vascular injury and enhanced microvascular permeability[21]. Systemic inflammatory response syndrome may also account for SFTS pathogenesis[22]. Similar pathogenic mechanisms between these viral hemorrhagic fevers may contribute to the severity of the disease. It is well known that HFRS and SFTS share similar clinical characteristics, such as thrombocytopenia, renal insufficiency, abnormal biochemical indicators, etc[23]. The simultaneous attack further increases the severity of the disease. In 2014, Korean scholar Sun Whan Park et al. [24] reported an HTNV/SFTSV co-infection case verified by serological tests, but molecular biology detection and virus isolation were not performed. In 2019, Liuwei et al. [25] conducted a retrospective analysis of 1546 febrile patients (603 HFRS and 943 SFTS patients), revealing that the co-infection rate of HTNV-SFTSV (0.6%, 9 of 1546 cases) was lower than predicted based on single HTNV and SFTSV infection rates. The results showed that the trend of co-infection between the two pathogens was low. The proportion of clinical features was not significantly higher in the HTNV-SFTSV co-infection group than in the HTNV or SFTSV infection groups alone, indicating that co-infection with both pathogens did not lead to more severe outcomes. This study confirmed for the first time that co-infection of HTNV and SFTSV caused severe HFRS, and the epidemic trend of HFRS had begun in Dandong. Therefore, co-infection of HFRS with other viral hemorrhagic fevers may lead to the emergence of critical cases.
Our findings identified a significant risk for severe HFRS in patients exposed to rodents, harboring the primary source of infection and host of HFRS. Mice contribute to disease spread via direct human exposure, exchange of virus-containing excreta (urine, feces, and saliva), or inhaled aerosols. The HTNV and SEOV positivity percentages were notably high in study areas. Rural environments often present poor housing and sanitary conditions, along with elevated rodent density during harvesting seasons, amplifying opportunities for contact with rodents, thus heightening the risk of direct or indirect transmission. Patients who have directly contact rodent history have high vigorous virus loads. Related research indicates severe/critical HFRS patients typically exhibit higher plasma virus levels in the early stages of the disease (5.90 vs. 5.03 log10 copies/mL, P =0.001), indicating a correlation between viral loads and disease severity[26].
The results also demonstrated that clinical features such as pharyngeal hyperemia, conjunctival congestion, abnormal white blood cells, urine protein, and IGM antibody positive significantly affected the severity of HFRS cases. Pharyngeal hyperemia and conjunctival congestion correlated with disrupted coagulation function in HFRS patients. Recent evidence suggested platelet counts may predict coagulation function and disease severity, thereby expanding prognostic capabilities and mitigating risk[27]. Moreover, renal dysfunction is a significant complication in HFRS as proteinuria appears. This urinary indicator reflects the severity of the disease[28]. Hantavirus infection engenders an inflammatory response. Cytokines associated with inflammation regulation positively correlate with white blood cell count and disease severity[29]. Several studies report HFRS shows acute kidney injury with transient proteinuria, with proteinuria reflecting the severity of the disease. Increased local heparanase activity in kidneys induced by hantavirus infection may disrupt endothelial glycocalyx, facilitating protein extravasation through the glomerular filtration barrier and leading to severe proteinuria[27]. In addition, this study revealed a higher severity rate for IGM positivity. Comparative investigations of cytokine levels in IGM-positive, -negative, and healthy groups identified elevated cytokines (IL-1ra, IL-12p70, IL-10, IP-10, IL-17, IL-2, and IL-6) in the IgM-positive group, suggesting disease progression[30]. Thus, specific clinical features contribute to the escalation of HFRS severity and impact initial clinical management.
This research corroborated that HTNV and SFTSV dual infection was determinant for severe HFRS cases combined molecular biology with virus isolation, emphasizing clinicians need to pay attention to the presence of multiple pathogens in HFRS severe case management. The severe patients primarily manifested HFRS symptoms but lacked SFTS respiratory and neurological manifestation. Normal or high counts of white cells indicate that SFTSV has not yet attacked the patient’s tissues and organs (Appendix). Hence, it was considered that HTNV is the primary pathogen of severe cases, and SFTSV is the synergistic pathogen, but there was the probability that the disease severity would exacerbate with viral load increase. HTNV and SFTSV destroy platelets in large quantities, making distinguishing what pathogen caused the platelet decline difficult. HTNV co-infections with different pathogens have been reported, such as an instance of HTNV co-infection with Dengue virus in Shenzhen in 2021. The patient had rodent contact, absent dengue fever epidemiology, and the source of infection was likely rodents[31]. Moreover, HTNV infection in elderly patients frequently accompanies other pre-existing conditions, predisposing to complications, critical-type incidence, and high death rate.
In this study, patient serum samples were evaluated using Vero and BHK cell lines, yielding two SFTSV isolates, but no HTNV. While the Vero-E6 cell line is typically employed for HTNV studies, the successful isolation rate remains subpar despite elevated viral burden during an early infection phase (febrile stage). There is an immediate need to identify a novel cell line exhibiting superior susceptibility to HTNV, facilitating its isolation. Moreover, numerous SFTSV isolates were obtained from ticks and SFTS patients in our institution over recent years, indicating ease of adaptation and proliferation on Vero cells. Simultaneously, it was observed that SFTSV proliferates rapidly upon inoculation with ticks harboring SFTSV and other viruses, potentially leading to complications.
According to the International Committee on Taxonomy of Viruses, there were 7 genera and 53 species of Hantaviridae with persistent novel species. HTNV is the primary pathogen of HFRS [1], a single-stranded negative RNA virus comprising three segments (L, M, S) that encode an RNA polymerase, glycoprotein, and nucleocapsid protein. In this study, the target gene for HFRS diagnosis is the glycoprotein sequence of segment M of the HTNV, which tends to undergo genetic mutations and modify virulence. Although the evolutionary analysis of the nucleic acid sequence of the fragment revealed that it aligned at approximately 95% identity with strains isolated in Jilin Province, Liaoning Province, it is insufficient to classify the virus as a novel subtype of Hantaan or evaluate the impact on pathogenicity. Limited sample sizes prevented complete gene amplification, requiring more sample collection and virus isolation for exploration. In addition, we isolated multiple SFTSV strains bearing genotype A in Liaoning Province, highly aligned with Anhui Province and Zhejiang Province, which is less virulent and has lower lethality in humans compared to other genotype strains prevalent in South Korea, Japan[14].