Hemorrhagic fever with renal syndrome caused by destruction of residential area of rodent in a construction site: epidemiological investigation

On November 15th, 2020, No. 986 hospital received a positive case of Hantavirus and reported that there was a possible outbreak of HFRS. In the following 3 days, 3 more HFRS patients were confirmed. As of December 2, a total of nine patients had been diagnosed with HFRS (Fig. 1). The site of the outbreak is in a new construction site, the construction action destructed the rodent’s residence. Between the two rows of dormitories where the patients lived there was a garbage station. In the north of the dormitory there was a tent, piled with Chinese cabbages and other vegetables for daily consumption. Rat tracks were observed inside the tent and near the garbage station (Fig. 2). The patients were classified into mild, moderate, and severe based on clinical classification of HFRS [10].

HFRS was diagnosed in all cases according to Enzyme-linked immunosorbent assay (ELISA)-based detection of anti-HV IgM in serum samples obtained from patients during acute phase of the disease [18]. The so-called acute phase of the disease is defined as the period of febrile, hypotensive, and oliguric phases [10, 18, 19]. In this study, ELISA was also used to detect the titers of serum IgG/M antibody over time in patients.

An epidemiological investigation was carried out after confirmation of the HFRS outbreak. HFRS-like syndrome (fever, hypotensive, proteinuria, and acute renal failure), activity tracks, clinical symptoms, and medical records of febrile patients residing at the construction site were retrospectively collected. The Hantavirus IgG antibody levels of all patients were monitored.

Epidemic prevention measures were carried out immediately after the outbreak. Rodent capture equipment, including 200 sticky rat boards, 800 traps and 30 cages, was set up immediately after the occurrence of the index case. The delivery of outdoor anticoagulant rodenticide was finished on November 16th. The garbage station was cleaned on November 12th-13th, the vegetables were removed from the tents and final disinfection was performed on the sites. A customized mouse baffle is installed in the canteen, and closed storage of materials is adopted. The local CDC strengthened the canteen daily cleaning, tableware/kitchenware strict disinfection, the management of water sanitation, the quarantine of personnel and vehicles. By the end of December, 1268 people had been vaccinated with inactivated bivalent (HTN + SEO type) HFRS vaccine made from hamster kidney cell culture (Changchun Institute of Biological Products, China). Experts were invited to carry out disease knowledge education, provide guidance on prevention and control work, educate personnel to maintain hygiene habits, and avoid panic. The temperatures of all staff were measured daily to check for fever or other uncomfortable conditions, and medical treatment was arranged in time when abnormal conditions occurred. The epidemic was basically brought under control about December 6 and eliminated about December 15th.

A. agrarius collected was sterilized with 75% alcohol, and the chest was dissected and a small lung tissue was taken out with a sterile scissors and tweezers. Appropriate amount of Trizol was added followed by tissue polishing. Magnetic bead method was used for nucleic acid extracting. HTNV-specific reverse transcription-polymerase chain reaction (RT-PCR) was conducted for viral RNA [20]. T25 VERO-E6 cells were cultured to a single layer, and the above-mentioned positive lung specimens were added to the cells for tiled adsorption [21]. Supernatant was removed and cell-sustaining fluid was added after 1.5 h. Viral CT values were detected after continuous cultivation of 21 days [22].

Viral RNA was extracted using RNeasy Plus Mini Kit (50) (QIAGEN 74134) [23]. Reverse transcription and amplification were performed using Ovation RNA-Seq System V2 (Tecan 7102-08). Amplification products were subjected to sequencing using Oxford Nanopore Gridion X5 instrument [24]. Library was constructed using ligation sequencing kit (SQK-LSK109) and the sequencing chip used in this study was FLO-MIN106D [25]. The consistency sequences were generated by mapping, with the known Hantaan virus whole genome as a reference sequence.

The nucleotide sequences of S, M, and L segments of hv03xa strain were determined from virus-infected lungs of A. agrarius. Sequences were aligned and compared with HTNV sequences available in GenBank using Mega (version 7.0) with default parameters [26]. For the phylogenetic analysis, the Maximum likelihood phylogenetic trees were generated, and topologies were evaluated by 1000 bootstrap replicates [26].

Alignments of Sequence sets to HTNV S, M, and L segments were analyzed using the Recombination Detection Program 4 (RDP4) software package [27]. RDP [28], GENECONV [29], BOOTSCAN [30], MaxChi [31], Chimaera [30], SISCAN [32] and 3SEQ [33] were used only on events with P values less than 0.05. The recombination event was confirmed if it satisfied by four or more methods when the P-value < 0.05 and the RDP recombination consensus score (RDPRCS) was > 0.6 [34]. The P-value < 0.05 with the RDPRCS between 0.4 and 0.6 indicated the possibility of the recombination event [34]. RDPRCS < 0.4 and P-value < 0.05 suggested the rejection of the recombination [34]. All parameters were left at the default settings of RDP4.

With a goal of determining the dynamics and duration of immunity produced after HTNV infection, sera collection was made 6 months and 12 months post-symptom onset. The anti-HV-IgG was tested by ELISA (ZC-M6404, Shanghai zcibio technology Co., Ltd) according to the manufacturer’s instructions. Briefly, the test was carried out by adding diluted serum onto a HV nucleocapsid protein antigen coated plate. After incubation for 30 min at 37 °C, wells were washed and incubated with 100 μl mouse anti-human IgG antibody labelled with horseradish peroxidase for 30 min at 37 °C. The IgG antibody captured by the antigen was detected by measuring the optical density (OD 450 nm) after adding the stop solution (0.2 M sulfuric acid). Each sample was repeated three times.

All participants signed an informed consent form prior to entering the study. The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki. All human and animal experimental protocols in this study were approved by the Ethics Committee of the PLA 63750 Hospital. All human and animal experimental procedures were carried out in accordance with relevant guidelines and regulations.

Nine HFRS patients developed symptoms consecutively from November 10th to 29th at Chang’an district, located in the southwest of Xi’an. All the patients were from the same construction site. The rodent residence in the surrounding place was destroyed because of the construction action. It is said that the rodent population density was significantly increased comparing with the same period in the past. Some Chinese cabbage was stored in a tent of their residence for their daily consumption (Fig. 2). On November 2nd, the index patient (a 22-year-old male) transported the cabbage from the tent to the kitchen with two other patients (P 2 and P6). They saw rats in the tent and in the garbage station near the tent (less than 50 m). The index patient developed a headache (November 10th) eight days after transporting the cabbage and vomited on the evening of November 13th. On the morning of November 14th, he developed a fever (39 °C) and was admitted to the hospital. Nucleic acid tests confirmed that he was not infected with COVID-19. On November 15th, he was diagnosed with hemorrhagic fever with renal syndrome (HFRS) and classified as moderate patient. P2 and P6 became ill on November 15th and November 25th, respectively. P3, P4, P7, and P8 lived in a dormitory near the garbage station (Fig. 2), and they frequently took out garbage to the garbage station before the onset of illness. P5 and P9 frequently visited the tent before they had symptoms. The timeline of disease prevalence was presented in Fig. 1.

The clinical characteristics of the patients were summarized in Additional file 1: Table S1. Based upon clinical classification of HFRS [10], of the 9 patients, 2 had no bilateral renal abnormalities, no hemorrhagic spots, and no bulbar conjunctival congestion, they were classified as mild. Four patients experienced moderate illness, they presented with symptoms such as oliguria, bulbar conjunctival congestion, and subcapsular effusion of both kidneys. Three patients presented with oliguria, bilateral bulbar conjunctival hyperemia and edema, visible hemorrhagic spots, hypotension, bilateral kidney pain, bilateral subcapsular effusion and other symptoms, they were defined as severe patients.

After confirmation of the outbreak on November 15th, demic prevention guarantee initiated a rodent eliminate program. Rodent capture was performed in the surrounding place. Meantime, publicity education efforts was also taken to control HFRS expansion. After that, the rodent density dropped obviously after rodent control efforts. Correspondingly, patient counts dramatically declined after December 1, approximately 10 days after the implementation of rodent control.

Strains information for sequence analysis in this study were shown in Table 1. The branches of the phylogenetic tree of Hantaan virus formed nine HTN clades (designated subtypes HTN 1–9) [17]. Phylogenetic analysis indicated that the hv03xa strain is likely a reassortment strain of HTNV. S segment nucleotide sequence of hv03xa strain was closely related to isolates of subtype HTN 4, including Xi’an isolates XAHu09066, CA09082007, CA10081206 and Shanxi isolate A16 [7], with identity of 98.94%, 98.88%, 98.53% and 98.70%, respectively. M segment of hv03xa strain was closely related to Guizhou strains Q37, Q10, CGHu3614, CGHu3612, CGRni1, CGAa31P9 and CGHu2 of subtype HTN 4 [17], with identity of 99.33%, 98.67%, 98.20%, 98.09%, 98.06%, 92.93% and 92.79%, respectively. L segment of hv03xa strain was closely related with strains originated from South Korea, including 76–118/PRO (84.27% identity), Aa04-722 (84.64% identity), Aa19-57 (84.63% identity) and Aa16-176 (84.62% identity), which were in the subtype HTN 7 clade (Fig. 3).

Different methods were employed to provide evidence that recombination could occur in HTNV. Potential recombination event was detected (Table 2). The hv03xa strain may have a recombinant S segment, in which nt 1–1664 originate from the Jiangsu strain A9 of HTNV 9, and nt 1665–1695 originates from Seoul virus strain ZT10 isolated from Zhejiang (Fig. 4). P-value of the analyses was from 3.74E-04 to 1.32E-10 and the RDP recombination consensus score was higher than 0.6 (0.679). The result indicated that hv03xa may have a partial S-segment exchange recombination. No recombination events were detected in the M or L segments of hv03xa strain.

The titer above 40 is considered a positive for HTNV-NP-specific IgM and IgG antibodies [9]. Overall, HFRS patients’ serum IgG antibody titers remain detectable and relatively stable one-year PSO (post–symptom onset) (Fig. 5). At 6 months PSO, the geometric mean titer (GMT) was 1248.09 (95% confidence interval [CI] 1067.39–1428.78). At one year after infection, IgG antibody titers were detected in all of 9 patients with a GMT of 1210.48 (95%CI 1085.20–1335.76), which has no significant difference with that of 6 months PSO (p < 0.05, Kruskal–Wallis test). The serum IgG antibody titers of patients with HTNV infection was not significantly decreased compared in 1 year later. The HTNV-NP-specific IgM antibody were negative at 6 months and 12 months in all serum samples.