Upolu virus and Aransas Bay virus, two presumptive bunyaviruses, are novel members of the family Orthomyxoviridae

Abstract

Emerging and zoonotic pathogens pose continuing threats to human health and ongoing challenges to diagnostics. As nucleic acid tests are playing increasingly prominent roles in diagnostics, the genetic characterization of molecularly uncharacterized agents is expected to significantly enhance detection and surveillance capabilities. We report the identification of two previously unrecognized members of the family Orthomyxoviridae, which includes the influenza viruses and the tick-transmitted Thogoto and Dhori viruses. We provide morphological, serologic, and genetic evidence that Upolu virus (UPOV) from Australia and Aransas Bay virus (ABV) from North America, both previously considered potential bunyaviruses based on electron microscopy and physicochemical features, are orthomyxoviruses instead. Their genomes show up to 68% nucleotide sequence identity to Thogoto virus (segment 2; ∼74% at the amino acid level) and a more distant relationship to Dhori virus, the two prototype viruses of the recognized species of the genus Thogotovirus. Despite sequence similarity, the coding potentials of UPOV and ABV differed from that of Thogoto virus, instead being like that of Dhori virus. Our findings suggest that the tick-transmitted viruses UPOV and ABV represent geographically distinct viruses in the genus Thogotovirus of the family Orthomyxoviridae that do not fit in the two currently recognized species of this genus.

Importance: Upolu virus (UPOV) and Aransas Bay virus (ABV) are shown to be orthomyxoviruses instead of bunyaviruses, as previously thought. Genetic characterization and adequate classification of agents are paramount in this molecular age to devise appropriate surveillance and diagnostics. Although more closely related to Thogoto virus by sequence, UPOV and ABV differ in their coding potentials by lacking a proposed pathogenicity factor. In this respect, they are similar to Dhori virus, which, despite the lack of a pathogenicity factor, can cause disease. These findings enable further studies into the evolution and pathogenicity of orthomyxoviruses.

FIG 1

Ultrastructure of Upolu virus (A) and Aransas Bay virus (B) in infected Vero E6 cell cultures. Bar = 100 nm.

FIG 2

Potential base pairing of UPOV and ABV segment terminal bases.

FIG 3

Phylogenetic analysis of deduced amino acid sequences of UPOV and ABV in comparison to those of other selected orthomyxoviruses, indicated by their GenBank accession number and abbreviation (UPOV, Upolu virus; ABV, Aransas Bay virus; JOSV, Jos virus; THOV, Thogoto virus; DHOV, Dhori virus; FLUCV, influenza C virus; FLUBV, influenza B virus; FLUAV, influenza A virus; QRFV, Quaranfil virus; JAV, Johnston Atoll virus). Neighbor-joining trees were constructed under a Jukes-Cantor model, running 1,000 pseudoreplicates; bootstrap values of >50% are indicated at the respective nodes; and scale bars indicate substitutions per site. (A) PB2 (segment 1); (B) PB1 (segment 2); (C) PA (segment 3); (D) GP (segment 4); (E) NP (segment 5); (F) M (segment 6).

FIG 4

Schematic of glycoprotein alignment including the tick-borne orthomyxoviruses Upolu virus (UPOV), Aransas Bay virus (ABV), Jos virus (JOSV), Thogoto virus (THOV), Dhori virus (DHOV), and Quaranfil virus (QRFV) as well as influenza A virus (FLUAV) and the insect Autographa californica multicapsid polyhedrosis virus (AcMNPV), showing the signal peptide (Signal); motifs of a potential fusion peptide cleavage site proposed for THOV (Fusion-pep); cysteine (C) residues conserved in all orthomyxoviruses or in the tick-borne orthomyxoviruses and AcMNPV , in tick-borne viruses and AcMNPV except DHOV , or in thogoto- and dhoriviruses or in thogotoviruses and AcMNPV ; conserved glycosylation sites surrounding position 183 (N₁₈₃GS/N₁₈₃GT; N₁₉₇VT in AcMNPV) and position 415/428 (NxT/S, including N_415/412/410XT/S in UPOV, ABV, and JOSV; N_{428/427/423/416}XT/S in UPOV, ABV, JOSV, and THOV; N₃₇₈NT in THOV; N₃₉₆HS in DHOV; N₄₂₂VS in QRFV; and N₃₈₄NS/N₄₂₆TT in AcMNPV); the trans-membrane anchor (TM); and amino acids of the cytoplasmic tail region (Cyt-tail).

FIG 5

Segment 6 coding strategies. (A) RNA extracts obtained from HEK 293 cells infected with Upolu virus (UPOV) or Jos virus (JOSV) (cellular RNA [cR]) or from DNase- and RNase-treated supernatants (genomic RNA [gR]). cDNA was amplified with primers located upstream of a potential splice region (p1) and downstream at an mRNA polyadenylation signal (p2) or at the segment terminal sequence (p3). M indicates molecular size markers. Only a single-size amplification product was observed with the UPOV template (bands 1 and 2), whereas differently sized products were generated with the JOSV template (bands 3 to 5). (B) Relevant sequences obtained from the respective bands shown in panel A. (C) Schematic of segment 6 coding strategies of Thogoto virus (THOV) and JOSV, and of Dhori virus (DHOV), Aransas Bay virus (ABV), and UPOV, indicating locations of primers p1, p2, and p3; ML or M ORF termination codons (ochre, opal, and amber); and splice sites.