Identication of a new chuvirus associated with febrile illness in China

Chuviruses belong to the genus Mivirus in the newly classied family Chuviridae, and their medical signicance remains undetermined 1,2 . Here, we identied a novel chuvirus, Nuomin virus (NOMV), from febrile patients in China. NOMV had a circular genome and presented enveloped spherical particles with a diameter of approximately 120–150 nm. Phylogenetically, NOMV formed a separate clade in the Chuviridae family, with 17.1–83.4% sequence identity to other chuviruses. NOMV infection was conrmed in 54 patients who had a tick-bitten history during 2017–2019, while both virus-specic antibodies and RNA were tested negative in healthy cohorts. The typical symptoms are characterized by fever and headache. Seroconversion or a four-fold elevation of antibody titers was observed in the available specimens of convalescent phase. NOMV was also detected in hard ticks, sheep, and cattle in northeastern China. Our ndings showed that a novel chuvirus is associated with human febrile illness in China, necessitating investigation of this emerging virus in the tick-endemic areas worldwide.


illness in China.
In May 2017, a 29-year-old male farmer in the Nuomin town in Inner Mongolia, China was admitted to the local hospital, due to the clinical symptoms of fever, headache, nausea, and vomiting. The patient had a tick bite before illness onset. He was infected with tick-borne encephalitis virus (TBEV) in 2014, and received inactivated TBEV vaccination in 2017. TBEV-speci c IgG antibodies were detected positive, but viral RNA was negative in the serum sample. Other tick-borne pathogens, including Alongshan virus, severe fever with thrombocytopenia syndrome virus (SFTSV), Babesia spp., Anaplasma spp., Rickettsia spp., Lyme disease spirochetes, were tested negative (Extended Data Table 1) [14][15][16][17][18][19][20] . To hunt the potential causative agent, the blood sample was used for metagenomic analysis, which resulted to several contigs annotated to Suffolk virus (SFKV, Supplemental Table S1) 3 .
Despite presence of numerous genomic sequences of new chuviruses in the last ve years, none has been isolated. We isolated the virus from the patient's blood sample by using Vero and BHK-21 cells, but no clear cytopathic effect was observed. Puri ed virions showed enveloped spherical particles, with a diameter of 120-150 nm under electronic microscopy (Fig. 1a). Viral particles in ultrathin sections could be observed in the cytoplasmic vacuoles of infected BHK-21 cells (Fig. 1b). We designated Nuomin virus (NOMV), and the disease was called Nuomin fever.
Real-time RT-PCR assay were established for quantitative detection of the NOMV (Extended Data Fig. 1) and evaluated the viral infectivity of multiple cell lines, including Vero, BHK-21, hepatocellular carcinoma cell (SMMC-7721), amniotic cell (WISH), and epithelial colorectal adenocarcinoma cell (Caco-2). The results showed that NOMV replicates more e ciently in BHK-21 and SMMC-7721 cells than in SMMC-7721 and WISH cells (Fig. 2). We later isolated ve additional strains from inpatients and one strain from Ixodes persulcatus. This was the rst isolation of virus species in the family Chuviridae.
The complete genome of NOMV strains was obtained based on metagenomic analysis (Extended Data   Fig. 2). The circular genome structure of NOMV was con rmed with around-the-genome RT-PCR. NOMV genome sequence shared high similarity with SFKV as compared with other chuviruses (Extended Data   Fig. 3). Interestingly, the head-to-tail sequences of NOMVs had a T-to-A/C substitution and an insertion of T. which shared 54.4% sequence identity to Suffolk virus (SFKV). Due to a lack of homology to proteins outside of the family Chuviridae, the potential function of VP4 remains unknown.
To determine the evolutionary relationships between NOMV and other chuviruses, phylogenetic trees were constructed with amino acid sequences of the L, G, and N proteins, respectively. NOMVs formed a separate clade from the viruses in the Chuviridae family and were clustered with viral members that have a circular genome; circular, bi-circular, or bi-segmented chuviruses may evolve from unsegmented viruses (Fig. 3). Notably, almost all chuviruses that contain a circular genome has been discovered in ticks (Fig. 3). of patients occurred during May to July (Extended Data Table 3).
We tested the serologic responses against NOMV by ELISA (Extended data Fig. 5), showing that all serum samples were IgM-positive in available serum samples from 14 patients at the acute period (AP), and IgG antibodies were gradually increased; 78.6% (11/14) of patients had a low level of neutralizing antibody at the AP, while high levels of neutralizing antibody were found in the CP specimens (Extended Data  Table 4). Seroconversion or at least 4 times as high as the titer in the AP specimens were detected in the six serum samples at the CP (Fig. 4).
Patients were given a combination of ribavirin and benzylpenicillin sodium. Ribavirin was given intravenously 0.5 g per day, and benzylpenicillin sodium was injected intramuscularly 4 million units per day. The clinical symptoms often resolved after treatment for 7 to 14 days.
We collected 2,147 hard ticks in the hilly and wooded regions where NOMV-infected patients were usually bitten. These ticks were divided into 148 pools and NOMV was detected by nested RT-PCR (Supplementary Table S5), showing a prevalence of 3.9%, i.e., 8.2% in Ixodes persulcatus, 7.8% in Ixodes crenulatus, 2.8% in Haemaphysalis conicinna, and 1.9% in Haemaphysalis longicornis. A higher prevalence was found in ticks in Heilongjiang (9.4%) than that in Inner Mongolia (4.0%) and Jilin (2.0%) provinces (p < 0.5) (Extended Data Table 7). We also detected viral RNA in the serum specimens of sheep and cattle in Inner Mongolia by real-time RT-PCR, revealing a prevalence of 21.7% (40/184) in sheep and 31.4% (79/252) in cattle. Partial RdRp genes of NOMV from ticks, cattle and sheep were obtained by using nested RT-PCR (Supplemental Table S5), and phylogenetic analysis showed that they were closely related to the human strains (Extended Data Fig. 6 and Table 8).
To explore the evolutionary origin of NOMVs, potential recombination was analyzed with the Recombination Detection Program 4.0 using available genomes. Several breakpoints were detected, which were also supported by similarity plot and bootscan analysis (Extended Data Fig. 7 and Table 9). Breakpoints were located at the nucleotides 526 and 6543 in L gene of H141, and at the nucleotides 6,656 and 9,037 in the G and partial N genes of H109, thus generating recombination fragments covering nucleotides 8567-10516 including partial G, N, and partial VP4 gene. Phylogenetic analysis suggested that human virus strains H141, H109, and H159 were the potential descendent of T43 detected in ticks.
The present study identi ed a new chuvirus in the family Chuviridae, the likely agent responsible for a febrile illness in China. Viral RNA positive in all patients, and viral protein seroconversion or at least 4 times as high as the titer observed in the available specimens at CP provides evidence of an association between the new chuvirus and the febrile illness. However, there are still many questions to be answered. Firstly, the association between the new chuvirus and the disease has not been con rmed by animal infection experiments to ful l the Koch's postulates. Secondly, though the virus has been detected in several tick species, their vectorial capacity needs to be further con rmed. Thirdly, it appears that NOMV cannot be transmitted from person to person, however, the virus should be closely monitored whether to evolve into a more virulent one. Fourthly, the pathogenesis mechanism should be elucidated and antiviral drugs and vaccines be developed for this emerging infectious disease. Lastly, the differential diagnosis of tick-borne diseases should include this emerging virus, whose public health signi cance makes it necessary to further investigate in the tick-endemic areas worldwide.

Study design and sample collection
We recruited patients who reported being bitten by ticks in the local hospital of Inner Mongolia in China during 2017-2019, and the blood specimens were collected to investigate potential tick-borne pathogens. A standardized questionnaire was used to collect data on tick exposure, demography, and medical history of each patient. The clinical symptoms and laboratory tests were obtained from the medical records.
We included the patients who were detected positive for viral RNA in the blood sample by real-time RT-PCR, and excluded the patients who had co-infection or infected with other pathogens. Two hundred healthy people in the same areas were included as controls.

Viral metagenomics
Viral metagenomics was conducted as described elsewhere 8 . Brie y, RNA was extracted from blood sample and reverse transcribed into double strand cDNA using the random primer 5´-GCCGGAGCTCTGCAGATATCNNNNNN-3´and a Klenow fragment. Sequence-independent single-primer ampli cation was used to amplify the double strand cDNA in a 50 µL reaction system that contained 25 µL Q5 high-delity Mix (NEB), 3 µL of the above double strand cDNA mixture, 3 µL primer (5´-GCCGGAGCTCTGCAGATATC-3´) and 19 µL ddH 2 O. The PCR products were puri ed for library construction and high-throughput sequencing in the Beijing Genome Institute (BGI, Shenzhen, China).

Genetic analysis
The complete genome of NOMV was obtained through nested RT-PCR with overlapping primers designed according to the metagenomic sequences as described in Extended data Fig. 2. The circular genomic form of NOMV was con rmed by around-the-genome nested RT-PCR 8 . Partial RdRp gene of NOMV from different hosts used for phylogenetic analysis were obtained by nested RT-PCR. All the primers used were listed in Supplemental Table S5-S6. PCR products were sequenced using the standard methods. Phylogenetic analyses were performed with the maximum likelihood method in the Molecular Evolutionary Genetics Analysis 5.0 (MEGA 5.0). The RdRp gene of typical viruses in the order Mononegavirales (132 strains) and 57 chuviruses were all used for phylogenetic analysis in the study (Supplemental Table S2-S3).
Virus isolation BHK-21 and Vero cells were used to isolate the new virus. They were grown in Dulbecco's modi ed eagle medium supplemented with 2% fetal bovine serum, 1000 units/ml penicillin and 1 mg/ml streptomycin antibiotics. The blood sample from the index patient was centrifuged at 12,000 g for 10 min; the supernatant was diluted 10 times and inoculated onto a con uent monolayer of BHK-21 or Vero cells for 1.5 h and replaced with DMEM supplemented with 1% FBS. The cells were cultured at 37°C in 5% CO 2 and monitored daily for cytopathic effect (CPE). Three blind passages, with a four-day interval between every passage, were conducted for each sample. The culture supernatant and cellular pellet of each passage were tested for the virus by real-time RT-PCR. Electron microscopy analysis was conducted as described previously 22 . Virus was quantitated by uorescent focus assay and viral titers were determined as uorescent focus units (FFU) per mL 23 .

Recombination analysis
Full-length genomic sequence of NOMV tick strain T43 (MW029970) was aligned with the available NMV strains from patients (H43, MW029971; H109, MW029972; H141, MW029973; H145, MW029974; H159, MW029975; H160, MW029976) using Clustal X. The potential recombinant events were preliminarily scanned using Recombinant Detection Program (RDP) v4.0 24 , and further investigated by similarity plot and bootscanning analyses in Simplot v3.5.1 25 . Phylogenetic origin of the major or minor parental regions of NOMV strains from patients were constructed from the essential ORFs of the major or minor parental regions of T43. ML phylogenetic trees of the three genome regions between the estimated breakpoints depend on the seven NOMV strains were constructed by MEGA 5.0 to understand the evolutionary relationship of NOMV 26 .

Serological analysis
Patients' serum was tested by ELISA using recombinant nucleoprotein (N). The N gene was cloned into pET-30a vector (Novagen) and its induced expression was conducted according to the routine methods. The N protein was puri ed and con rmed with an anti-His-tag antibody. NOMV-speci c IgM and IgG antibodies were detected in patients as described elsewhere 27 . Brie y, the coating antigen was the expressed N protein (5 ng/µL), serial dilutions of patient serum (starting at 1:10) by a factor of two were incubated in an indirect ELISA. The cutoff value of the reaction was calculated as the mean OD of 200 healthy blood donors' sera OD values plus two standard deviations. A sample that showed higher OD value than cutoff line with a titer of ≥ 1:10 was considered NOMV serological IgM or IgG positive.
Neutralizing antibodies for NOMV in serum samples were tested by microneutralization test (MNT), serial dilutions of serum samples (starting at 1:10) by a factor of two were mixed with an equal volume of approximately 100 FFUs NOMV and incubated at 37°C for 1.5 h. The mixture was then added to a 96-well plate in quadruplicate, which was seeded with 1× 10 4 BHK-21 cells 12 h before infection. After 3 days incubation, viral infection was detected by IFA using serum samples from patients with laboratorycon rmed infection. The end-point titer was de ned as the reciprocal of the highest dilution of serum that prevented infection.
Immuno uorescence assay (IFA) was used to detect the infection of NOMV in different cell lines. Vero, BHK-21, SMMC-7721, WISH, and Caco-2 cells were infected with NOMV at a MOI of 5 in triplicate for 48 h.
After xed with methanol and acetone (1:1), the cells were washed with 1×PBS three times and blocked with 5% BSA for 1 h. The convalescent serum specimens with a dilution of 1:20 were added and incubated for 1 h and probed with uorescein isothiocyanate-conjugated goat anti-human IgG for another hour. The cellular nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min.

Data analysis
The complete sequences of viruses were analyzed by ORF nder (https://www.ncbi.nlm.nih.gov/or nder) to nd putative viral proteins 28      Serological investigation of NOMV patients. Virus-speci c IgM (a) and IgG (b) antibodies in six patients were detected by the N protein-based ELISA. The red rhombuses and blue dots indicate the OD values of each patient in the acute period (AP) and convalescent period (CP), respectively (More information can be found in Extended Data Table 4). The black dotted line matches the OD value of the same patient in AP and CP. The cutoff value was set up as 0.38 for IgM test and 0.40 for IgG test according to healthy controls.