Metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Henan Province, China: discovery of a new bunyavirus

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.

Figure 1. Phylogenetic analysis of novel bunyavirus proteins.

Phylogenetic trees were generated by comparing the translated amino acid sequences of individual sequence reads to the corresponding sequences from known bunyaviruses using a neighbor-joining method. The horizontal-line distance represents the number of sites at which the two compared sequences are different. Bootstrap values deduced from 1000 replicates. A: C361 fragment (Accession number HQ412604). B: FTLS bunyavirus (Henan isolate), Segment L (Accession number HQ642766). C: FTLS bunyavirus (Henan isolate), Segment M (Accession number HQ642767). D: FTLS bunyavirus (Henan isolate), Segment S (Np) (Accession number HQ642768). E: FTLS bunyavirus (Henan isolate), Segment S (Ns) (Accession number HQ642768).

Figure 2. Vero E6 cells inoculated with acute serum specimens from patients with FTLS.

The typical early CPE seen with novel bunyavirus isolates from patients with FTLS is shown in part A (×40). Mock-inoculated Vero cells are shown in part B (×40). Infected Vero cells reacted with the serum of a convalescent FTLS patient in an indirect IFA in part C (×400).

Figure 3. Thin-section electron microscopy of novel FTLS-associated bunyavirus Grown in Vero E6 Cells.