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].