Metagenomic Analysis of Fever, Thrombocytopenia and Leukopenia Syndrome (FTLS) in Henan Province, China: Discovery of a New Bunyavirus

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Abstract
Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here, we used an unbiased metagenomic approach to screen and survey for microbes possibly associated with FTLS. BLASTx analysis of deduced protein sequences revealed that a novel bunyavirus (36% identity to Tehran virus, accession: HQ412604) was present only in sera from FTLS patients. A phylogenetic analysis further showed that, although closely related to Uukuniemi virus of the Phlebovirus genus, this virus was distinct. The candidate virus was examined for association with FTLS among samples collected from Henan province during 2007–2010. RT-PCR, viral cultures, and a seroepidemiologic survey were undertaken. RT-PCR results showed that 223 of 285 (78.24%) acute-phase serum samples contained viral RNA. Of 95 patients for whom paired acute and convalescent sera were available, 73 had serologic evidence of infection, with 52 seroconversions and 21 exhibiting a 4-fold increase in antibody titer to the virus. The new virus was isolated from patient acute-phase serum samples and named Henan Fever Virus (HNF virus). Whole-genome sequencing confirmed that the virus was a novel bunyavirus with genetic similarity to known bunyaviruses, and was most closely related to the Uukuniemi virus (34%, 24%, and 29% of maximum identity, respectively, for segment L, M, S at maximum query coverage). After the release of the GenBank sequences of SFTSV, we found that they were nearly identical (>99% identity). These results show that the novel bunyavirus (HNF virus) is strongly correlated with FTLS. Initially in 2007, and again between 2008 and 2010, cases of a life-threatening disease with sudden fever, thrombocytopenia, and leukopenia were reported in Henan Province, China. Patient reports of tick bites suggested that this disease could be infectious or tick-transmitted. Many patients were provisionally diagnosed with human granulocytic anaplasmosis (HGA). However, only 24 of 285 (8%) had objective evidence of HGA, suggesting that other pathogens likely contributed to fever, thrombocytopenia and leukopenia syndrome (FTLS). Illumina sequencing was used for direct detection in clinical samples of pathogens possibly associated with FTLS. A novel bunyavirus was found only in samples from FTLS patients. Further epidemiologic and laboratory investigation confirmed that the novel bunyavirus was associated with FTLS. The results illustrate that metagenomic analysis is a powerful method for the discovery of novel pathogenic agents. Combined with epidemiologic investigation, it could assist in rapid diagnosis of unknown diseases and distinguish them from other diseases with similar symptoms caused by known pathogens.