Characterization of the Bhanja serogroup viruses (Bunyaviridae): a novel species of the genus Phlebovirus and its relationship with other emerging tick-borne phleboviruses

Abstract

Bhanja virus (BHAV) and its antigenically close relatives Forecariah virus (FORV), Kismayo virus (KISV), and Palma virus (PALV) are thought to be members of the family Bunyaviridae, but they have not been assigned to a genus or species. Despite their broad geographical distribution and reports that BHAV causes sporadic cases of febrile illness and encephalitis in humans, the public health importance of the Bhanja serogroup viruses remains unclear, due in part to the lack of sequence and biochemical information for the virus proteins. In order to better define the molecular characteristics of this group, we determined the full-length sequences of the L, M, and S genome segments of multiple isolates of BHAV as well as FORV and PALV. The genome structures of these Bhanja viruses are similar to those of viruses belonging to the genus Phlebovirus. Functional domains and amino acid motifs in the viral proteins that are conserved among other known phleboviruses were also identified in proteins of the BHAV group. Phylogenetic and serological analyses revealed that the BHAVs are most closely related to the novel emerging tick-borne phleboviruses severe fever with thrombocytopenia syndrome virus and Heartland virus, which have recently been implicated as causing severe acute febrile illnesses associated with thrombocytopenia in humans in China and the United States. Our results indicate that the Bhanja serogroup viruses constitute a single novel species in the genus Phlebovirus. The results of this study should facilitate epidemiological surveillance for other, similar tick-borne phleboviruses that may represent unrecognized causes of febrile illness in humans.

Fig 1

Geographic distribution of Bhanja group viruses. The countries or regions where Bhanja group viruses have been isolated or where specific antibodies to Bhanja group viruses have been detected in animals or humans are shaded. The geographic locations where the viruses used in the present study were first isolated are indicated by black dots. The names and strain numbers of the isolates are given.

Fig 2

Growth of Bhanja viruses in cell lines. (A and B) The growth of Bhanja virus (BHAV) IbAr2709 in DH82 cells was confirmed by electron microscopy. (A) Virions in an intracellular vacuole; (B) inclusion bodies. Bars, 100 nm. (C) Three isolates of BHAV and an isolate of Palma virus (PALV) were tested for growth in Vero E6 and DH82 cells. The cells were infected at an MOI of 0.05, and the supernatants of the cells at each time point were titrated by a plaque assay using Vero E6 cells. The experiments were performed in triplicate. Error bars indicate the standard errors of the means.

Fig 3

Genome structure of Bhanja virus. (A) Schematic diagram of the coding strategy of Bhanja virus. BHAV has three genome segment RNAs with different lengths (nucleotide sequence lengths are shown on the right). The largest segment (L) encodes an RNA-dependent RNA polymerase (RdRp), and the second largest segment (M) encodes a glycoprotein (GP). The smallest RNA segment (S) encodes a nonstructural protein (NSs) and a nucleocapsid protein (N) in opposing directions. The amino acid (aa) sequence length of each protein is indicated above the corresponding open reading frame (shown as an open arrow). (B) Terminal conserved complementary sequences that constitute a “panhandle structure” in each genome segment. The upper strand in each alignment starts from the 3′ end of the genome, while the lower strand starts from the 5′ end. Vertical lines between the two strands indicate complementary nucleotides in the 3′ and 5′ ends. The conserved 3′ and 5′ terminal nucleotides of each genome segment are shaded. SFTSV, severe fever with thrombocytopenia syndrome virus; UUKV, Uukuniemi virus; RVFV, Rift Valley fever virus; GOUV, Gouleako virus (55). (C) The predicted secondary structure of the intergenic region between the two ORFs of the S segment RNA was calculated using CLC Main Workbench (CLC bio). Each number indicates the nucleotide position from the 3′ end of the virus genome.

Fig 4

Comparison of the glycoproteins among tick-borne phleboviruses. (Top) Schematic diagrams of the predicted structure of GP for Bhanja virus IG690 (BHAV), severe fever with thrombocytopenia syndrome virus (SFTSV), and Uukuniemi virus (UUKV). The length of the total amino acid (aa) sequence for each protein is given on the right. Signal sequences (shaded rectangles) were predicted with SignalP 4.0, and transmembrane domains (filled rectangles) were predicted with MEMSAT3. Each region or domain starts from the amino acid position indicated above the rectangle and ends at the position indicated below the rectangle. N-linked glycosylation sites were predicted with NetNGlyc, version 1.0, and are indicated by the letter “Y” above each diagram. (Bottom) Alignment of the amino acid sequences of the areas around the transmembrane region of Gn (left) and the cytoplasmic tail of Gc (right) in tick-borne phleboviruses. Numbers above the alignment indicate the amino acid position in the BHAV IG690 GP. FORV, Forecariah virus; PALV, Palma virus. (Left) The deduced transmembrane domains are boxed, and the Golgi retention motif of UUKV GP (47) is indicated by black dots under the alignment. (Right) The conserved lysine at the third amino acid position from the C terminus of GP is shaded.

Fig 5

Comparison of amino acid sequences of the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) among tick-borne phleboviruses. (A) The amino acid residues of the so-called ‘polymerase module’ of RdRp were compared among tick-borne phleboviruses: Bhanja virus (BHAV) IG690, Severe Fever and Thrombocytopenia virus (SFTSV), and Uukuniemi virus (UUKV). Amino acids conserved among negative-stranded RNA viruses have been highlighted in gray and ones conserved only among bunyaviruses have been underlined (53). Numbers above the aligned sequences indicate the amino acid positions in the BHAV IG690 RdRp. In the RNA-dependent RNA polymerases (RdRp) of negative-stranded RNA viruses, the polymerase modules are conserved among different virus families, including phlebovirus, suggesting that the modules represent the amino acids crucial for polymerase function. (B) Alignment of the RNA-binding region of the N protein. The asterisks on the bottom of the sequences indicate the amino acids crucial for the ability of N to bind viral RNAs, which are conserved among phleboviruses (54). The conserved amino acids among tick-borne phleboviruses are indicated by gray boxes. Numbers above the aligned sequences indicate the amino acid positions in the BHAV IG690 N protein.

Fig 6

Phylogenetic relationships among phleboviruses. Maximum likelihood trees were constructed with the GTR + Γ + I model based on the nucleotide sequences of the L segment RNA (A), the M segment RNA (B), and the S segment RNA (C). Numbers on the trees represent bootstrap values of 1,000 replicates. Abbreviations: BHAV, Bhanja virus, FORV, Forecariah virus; PALV, Palma virus; SFTSV, severe fever with thrombocytopenia syndrome virus; RVFV, Rift Valley fever virus.

Fig 7

Phylogenetic relationships among phlebovirus proteins. Maximum likelihood trees based on the amino acid sequences of each viral protein were constructed using the PROTGAMMA model. (A) RNA-dependent RNA polymerase (RdRp), encoded by the L segment; (B) glycoprotein, encoded by the M segment; (C) nucleocapsid protein (N), encoded by the S segment; (D) nonstructural protein (NSs), also encoded by the S segment. Numbers on the trees represent bootstrap values of 1,000 replicates. Abbreviations of taxa: BHAV, Bhanja virus; FORV, Forecariah virus; PALV, Palma virus; SFTSV, severe fever with thrombocytopenia syndrome virus; RVFV, Rift Valley fever virus.