Chuviruses, a group of single-stranded negative sense RNA viruses, have been discovered in arthropods, snakes, fishes, and nematodes, with the unsegmented, bi-segmented, circular, and bi-circular genomic structures that encodes the large protein (L), glycoprotein (G), nucleoprotein (N), and an uncharacterized viral protein (VP)3–7. Chuviruses are genetically situated between segmented and unsegmented negative-sense RNA viruses4. Most chuviruses were discovered in the middle and lower reaches of the Yangzi River region, which was called Chu in ancient China6,8. Chuviruses have also been found in United States of America3, Brazil9, Australia10, and other countries11–13. However, their relevance to public health remains unclear. Here, we identified a new chuvirus, Nuomin virus (NOMV), that is associated with human febrile 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-specific 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)14–20. 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 five 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. Purified 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 efficiently in BHK-21 and SMMC-7721 cells than in SMMC-7721 and WISH cells (Fig. 2). We later isolated five additional strains from inpatients and one strain from Ixodes persulcatus. This was the first 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 confirmed 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. The length of NOMV genome was 10,900 bp, and contained 4 open reading frames (ORFs) encoding the large protein (L, 6516 nt), glycoprotein (G, 2001 nt), nucleoprotein (N, 1278 nt), and an unknown viral protein (VP4, 324 nt), respectively. ORF1 encoded a 2171-aa large protein (L), which had 17.1–83.4% identity to other chuviruses (Extended data Table 2), and characterized by the conserved domains responsible for viral RNA replication (Extended Data Fig. 3)6. ORF2 encoded a putative 666-aa glycoprotein (G), with 36.4–89.6% similarity to other chuviruses. ORF3 encoded a putative 425-aa nucleoprotein (N), with 20.1–93.5% identity to other chuviruses. ORF4 encoded a 107-aa protein (VP4), 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).
During 2017–2019, a total of 54 patients were confirmed NOMV infection by real-time RT-PCR. Of these patients, 14 were found in 2017, 20 in both 2018 and 2019; 34 were from Inner Mongolia, 18 were from Heilongjiang, and one each from Jilin and Liaoning (Extended Data Fig. 4); 66.7% (36/54) of patients were men and their ages ranged from 22 to 69 years old, with 63.0% (34/54) between 40 and 60 years old; 48 (88.9%) were field workers, and all had clear history of tick bites before the onset of illness; 83.3% of patients occurred during May to July (Extended Data Table 3).
The median time from tick bite to illness onset (incubation period, IP) was 5 days (IQR, 2–9 days), from tick bite to hospital admission was 8 days (IQR, 6–14 days), and from tick bite to discharge was 20 days (IQR, 17–27 days).
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).
NOMV caused non-specific manifestations including fever and headache. Other clinical findings may include depression, dizziness, fatigue, myalgia, arthralgia, nausea, cough, rash, or petechiae (Extended Data Table 5). Biochemical analysis of blood showed elevated monocytes (30.2%), neutrophils (24.5%), and decreased lymphocytes (28.3%). The most common abnormal biochemical indicators included elevated high-sensitivity C-reactive protein (66.7%) and decreased apolipoprotein AI (75.9%). Liver injury was shown in approximately 30% of patients, and myocardial injury in approximately 10%, as indicated by increased serum aspartate aminotransferase (44.2%), alanine aminotransferase (32.7%), lactate dehydrogenase (26.3%), and creatine kinase (10%) (Extended Data Table 6).
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 identified 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 confirmed by animal infection experiments to fulfil the Koch’s postulates. Secondly, though the virus has been detected in several tick species, their vectorial capacity needs to be further confirmed. 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 significance makes it necessary to further investigate in the tick-endemic areas worldwide.