Gene duplication and phylogeography of North American members of the Hart Park serogroup of avian rhabdoviruses

Abstract

Flanders virus (FLAV) and Hart Park virus (HPV) are rhabdoviruses that circulate in mosquito-bird cycles in the eastern and western United States, respectively, and constitute the only two North American representatives of the Hart Park serogroup. Previously, it was suggested that FLAV is unique among the rhabdoviruses in that it contains two pseudogenes located between the P and M genes, while the cognate sequence for HPV has been lacking. Herein, we demonstrate that FLAV and HPV do not contain pseudogenes in this region, but encode three small functional proteins designated as U1-U3 that apparently arose by gene duplication. To further investigate the U1-U3 region, we conducted the first large-scale evolutionary analysis of a member of the Hart Park serogroup by analyzing over 100 spatially and temporally distinct FLAV isolates. Our phylogeographic analysis demonstrates that although FLAV appears to be slowly evolving, phylogenetically divergent lineages co-circulate sympatrically.

Keywords: Bird-associated arbovirus; Coupled translation; Flanders virus; Gene duplication; Hart Park serogroup; Hart Park virus; Rhabdovirus; SH protein; U1–U3 proteins.

Fig. 1

SDS-PAGE and immunoblot analysis of FLAV-infected Vero cell lysates. The membrane was probed with FLAV-specific mouse hyperimmune ascites fluid and a goat anti-mouse IgG-HRP conjugate. The molecular weights (kDa) of the individual proteins in the ladder are indicated and the tentative FLAV protein designations of the immunoreactive bands are shown. See text for details. Individual lanes are as follows: (A) FLAV-infected Vero cells, day 3 post-infection; (B) mock-infected Vero cells; (C) protein ladder (20–150 kDa).

Fig. 2

Comparison of the termination–reinitiation sequence and termination upstream ribosome-binding site (TURBS) motifs that are essential for coupled translation in select RNA viruses versus the cognate region in the FLAV G/SH ORF junction. The pentanucleotide termination–reinitiation sequence (UAAUG) is shown in bold italics, the 18S rRNA complementary region (motif 1) is in dark blue (with mismatches between the viruses shown in royal blue), and the two base-pairing motifs (motif 2 and 2^⁎) postulated to form RNA secondary structures are shown in grey (Powell, 2010). The viruses (and their GenBank accession numbers) used in the alignment were: FLAV 61-7484 (KF028661), Norovirus Hu/GII.4/CHDC4871/1977/US (FJ537138), Norwalk-like virus SW/NV/swine43/JP (AB126320), and Influenza B virus B/Rochester/02/2001 (KC892131). The two ORFs/proteins involved in coupled expression for each virus are indicated.

Fig. 3

Newly proposed genomic configuration of FLAV demonstrating the U1–U3 region between the P and M genes and the SH ORF between the G and L genes. The genomic organization of the related Wongabel virus (WONV) is shown for comparison.

Fig. 4

Alignment of the U1–U3 proteins of the prototype isolate of FLAV (61-7484) showing amino acid identity indicative of gene duplication. Residues of identity are highlighted in blue. Asterisks, colons, and periods indicate identical, conserved, and semi-conserved residues, respectively, among the three proteins. Tryptic peptides of U1 and U3 identified by mass spectrometry are shown in bold italic.

Fig. 5

Evolutionary relationships among 103 U1 nucleotide sequences of FLAV depicting the two main viral lineages (A) and (B). To illustrate the sympatric co-circulation of the two lineages, those viruses sampled from Lowndes County, Georgia, are shown in bold italics. All horizontal branches are scaled according to the number of nucleotide substitutions per site, and bootstrap support values are shown for key nodes.