Isolation and characterization of genetic variants of Orthohantavirus hantanense from clinical cases of HFRS in Jiangxi Province, China

Abstract

Background: Hemorrhagic fever with renal syndrome (HFRS) is a severe public health problem in Jiangxi province, China. Previous studies reported genetic variants of Orthohantavirus hantanense (Hantaan virus, HTNV) in rodents in this area. However, the relationship between HTNV variants and human infection needs to be confirmed. This study aimed to identify the HTNV variants in patients and to understand the clinical characteristics of HFRS caused by these variants.

Methods: Samples were collected from hospitalized suspected cases of HFRS during the acute phase. HFRS cases were confirmed using quantitative real-time RT-PCR. Peripheral blood mononuclear cells (PBMC) from patients with HFRS were inoculated into Vero-E6 cells for viral isolation. The genomic sequences of HTNV from patients were obtained by amplicon-based next-generation sequencing. A retrospective analysis was conducted on the clinical characteristics of the patients.

Results: HTNV RNA was detected in 53 of 183 suspected HFRS patients. Thirteen HTNVs were isolated from 32 PBMCs of HFRS cases. Whole genome sequences of 14 HTNVs were obtained, including 13 isolates in cell culture from 13 patients, and one from plasma of the fatal case which was not isolated successfully in cell culture. Genetic analysis revealed that the HTNV sequence from the 14 patients showed significant variations in nucleotide and amino acid to the HTNV strains found in other areas. Fever (100%, 53/53), thrombocytopenia (100%, 53/53), increased serum aspartate aminotransferase (100%, 53/53), and increased lactate dehydrogenase (96.2%, 51/53) were the most common characteristics. Severe acute kidney injury was observed in 13.2% (7/53) of cases. Clinical symptoms, such as pain, petechiae, and gastrointestinal or respiratory symptoms were uncommon.

Conclusion: The HTNV genetic variants cause human infections in Jiangxi. The clinical symptoms of HFRS caused by the HTNV genetic variant during the acute phase are atypical. In addition to renal dysfunction, attention should be paid to the common liver injuries caused by these genetic variants.

Fig 1. One-step amplicon-based NGS in this study.

A. Schematic diagram of HTNV genome covered by amplicons produced by 10 pairs of primers. B. Amplicons obtained using the primers were validated through electrophoresis. C. The workflow of one-step amplicon-based NGS.

Fig 2. Flow diagram of the study.

“*”: Among these 32 patients, virus isolation was performed simultaneously using plasma (isolate n = 0) and PBMCs (isolates n = 3) from 7 paitens, and virus isolation was performed using only PBMC from 25 patients (isolates n = 10).

Fig 3. The IFA images of HTNV-infected Vero-E6 cell and Vero-E6 cells control.

HTNV-infected Vero-E6 cells show specific bright green fluorescent dots indicating the virus was isolated successfully (A), while the control Vero-E6 cells show no bright green fluorescent dots (B).

Fig 4. TEM images of HTNV particles (isolate JXGAHu98/2021) in Vero-E6 Cell.

A vesicle of the HTNV-infected cell was magnified, virions were observed inside the vesicles, and arrows pointed to the HTNV particles. “N”: the nuclear of the cell.

Fig 5. Phylogenetic tree of the orthohantavirus using the complete M sequence.

The scale bars indicate the number of nucleotide substitutions per site. Strains labeled in red represent HTNVs from 14 patients in this study and those in blue represent HTNV isolates from rodents in Jiangxi but not in this study. PUUV, Orthohantavirus puumalaense. SEOV: Orthohantavirus seoulense.

Fig 6. The proportion of patients with elevated liver and renal markers (n = 53).

AST, LDH, ALT, ALP, GGT, and DB were liver function indicators, and proteinuria, BUN, and SCR were renal function indicators. The ULN levels of AST, LDH, ALT, ALP, GGT, DB, BUN, SCR, and proteinuria are 40 U/L, 240U/L, 50 U/L, 135 U/L, 60 U/L, 6 μmol/L, 120 μmol/L, 7.1 mmol/L, and 0.15 g/L, respectively.