Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus

Abstract

Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, MA, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here, we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein that was demonstrated to have fusion activity; this also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined.

Importance: The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here, we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.

FIG 1

Morbidity and mortality of common eiders in association with natural WFBV infection in Cape Cod, MA, USA. (A) Morbid female eider displaying reversed head posture and extreme lethargy, common clinical signs in WFBV-infected birds. (B) Multi-institutional disease investigation of a mass die-off during 2006. Hundreds to thousands of dead eiders have been documented during such cyclic outbreaks, which generally appear annually during the fall. Inset, WFBV-infected birds such as this male eider (sexes are dimorphic) exhibit listlessness and recumbency and are easily captured. (Photo B courtesy of Jim Canavan, Woods Hole Oceanographic Institution.)

FIG 2

Gross and histopathological lesions associated with natural WFBV infection in common eiders. (A) Coelomic cavity of a WFBV-infected eider. The liver contains coalescing to focally extensive (arrowheads) and multifocal (arrow) white to tan lesions on the serosal and cut surfaces. Scale bar = 1 cm. (B) Microscopic view of a liver lesion, characterized by variably sized areas of acute hepatocellular necrosis and hemorrhage. Scale bar = 100 μm. (C) Colocalization of WFBV antigen and pathological lesions. Eiders that were observed showing clinical signs of disease, and from which virus was isolated, were tested for viral antigen by immunohistochemistry using anti-WFBV mouse hyperimmune ascites fluid. Viral antigen was detected in hepatocellular lesions using DAB chromogen (shown as reddish-brown precipitate). Scale bar = 100 μm. (D) Acanthocephalan infection in the intestinal tract of a common eider infected with WFBV. The intestine has been cut transversely to display the yellow spiny-headed worms of the phylum Acanthocephala that are ubiquitous enteric parasites of common eiders. Note the attachment of the proboscis to the intestinal wall (arrowheads) and associated inflammation. Scale bar = 0.5 cm.

FIG 3

Transmission electron microscopy of WFBV. (A) Ultrathin-section micrograph of two virions budding from the surface of infected Vero E6 cells. Scale bar = 100 nm. (B) Transversely sectioned virion (∼100 nm in diameter) from infected BHK cells, apparently showing seven ribonucleoprotein complexes. (C) Virions negatively stained with 2% potassium phosphotungstate, pH 7.0, with protruding hemagglutinin (HA) glycoproteins visible on the surface. Magnification = ×60,000. Scale bar = 100 nm.

FIG 4

Segmental configuration of the WFBV genome and identification of a new orthomyxovirus gene (VP7). All seven identified RNA segments of the WFBV genome are shown in positive-sense orientation in decreasing size from top to bottom. For each segment, the 5′ and 3′ terminal noncoding regions (NCRs) are shown as thin black lines, and each open reading frame (ORF) is shown as a thick gray bar. The nucleotides encompassing the ORF of each gene are shown (in black) on the left side of each gray bar, while the size of the protein product (in amino acids) produced from the ORF is shown (in blue) on the right. The total length of each RNA segment (NCRs and ORF) is shown on the very far right. Note that the smallest segment (segment 7) is 519 nt long and encodes a previously undescribed protein of unknown function (tentatively designated VP7). The complete genome of WFBV has been deposited in GenBank under the accession numbers KM114304 to KM114310.

FIG 5

Structural protein analysis of WFBV. Purified virions were analyzed by SDS-PAGE followed by nanoscale high-performance liquid chromatography coupled to tandem mass spectrometry (nano HPLC-MS/MS). All discrete bands in the gel were excised and subjected to tryptic digestion, and peptides were identified by nano HPLC-MS/MS (see Fig. S1 in the supplemental material for peptide analysis). The molecular masses (kDa) of the individual proteins in the ladder (right) are indicated for cross-reference. Note that the putative M protein (predicted to be 29.7 kDa) is abundantly present in virions. Although peptides corresponding to PA, PB1, and PB2 were detected in visible bands (lower asterisk), discrete bands for these polymerase proteins were not visible, possibly owing to their low abundance within virus particles relative to other structural proteins. The majority protein present in the high-molecular-mass bands (two asterisks) was syndecan 4, a cellular transmembrane proteoglycan.

FIG 6

Fusion activity of the WFBV hemagglutinin (HA) protein and the predicted molecular model of the HA monomer showing its structural relationship to the postfusion conformation of group I alphabaculovirus gp64. (A) The WFBV HA protein was modeled using the Autographa californica nucleopolyhedrovirus (AcMNPV) gp64 protein as the template (PDB 3DUZ). The highlighted domains of the WFBV HA (I to V) are based on the homologous domains of the baculovirus gp64. Disordered regions of the AcMNPV gp64 (denoted as dots) (17) are shown in black in WFBV. Note the location of the fusion loops in domain Ia and the highlighted N and C termini. (B) Comparison of the fusion loops of AcMNPV gp64 and WFBV HA. Residues Y75 to T86 in loop 1 and N149 to H156 in loop 2 are critical for fusion activity and receptor binding (22), with three histidines in loop 2 (H152, H155, and H156) important for pore expansion (63). Conserved residues are highlighted by stick representations, with differences between the two viruses shown in marine. Residues outside the Y75-T86 and N149-H156 regions are shown in dark gray. (C to H) pH-dependent cell-to-cell fusion in virus-infected and HA-transfected cells. WFBV-infected (10-280-G) Vero cells (C) were incubated in MEM (pH 4.0) for 10 min and then returned to physiological pH (pH 7.4) (D). Note the formation of various-sized syncytia (arrowheads) in WFBV-infected cells 24 h after low-pH treatment, as shown in panel D. In transfection experiments, neither Vero cells transfected with empty vector and low-pH-treated (E) or HA-transfected cells at physiological pH (F) demonstrated cell-to-cell fusion. However, discrete syncytium formation was evident within 4 h of low-pH treatment in HA-transfected cells (G), as demonstrated by the congregation of nuclei (white arrowheads) and development of large expanses of cytoplasm (black arrowhead). A closeup photo of HA-transfected cells 24 h after low-pH treatment (H) clearly demonstrated large classical multinucleated cells (white arrowhead), along with late-stage detachment of syncytia (black arrowhead). Scale bars = 200 μm.

FIG 7

Evolutionary relationships of WFBV and other orthomyxoviruses. (A) Phylogeny based on the PB1 amino acid sequences of representative members of five genera of Orthomyxoviridae is shown. The tree is drawn to a scale of amino acid substitutions per site (with all ambiguously aligned positions removed), and all bootstrap values of >70% are shown, along with the bootstrap value relating to WFBV. The tree is midpoint rooted for purposes of clarity only. Viruses are shown as designated or proposed ICTV abbreviation/host/location/strain/year. Viruses (and their GenBank accession numbers) are as follows (note only ICTV-approved species are italicized): ABV, Aransas Bay virus RRML65660-8 (KC506163); DHOV, Dhori virus I-611313 (P27153); FLUAV, Influenza A virus Netherlands/1 (ACR58714), Tibet/8 (ADG59444), Guatemala/060 (AFC35436), and Delaware/127 (ACZ45637); FLUBV, Influenza B virus Singapore/222 (AAF06873), Oklahoma/347 (ACN32565), and Brazil/975 (ABL84349); FLUCV, Influenza C virus Ann Arbor/1 (YP_089653), Johannesburg/1 (Q9IMP4), and Oklahoma/1334 (AFJ19019); JAV, Johnston Atoll virus LBJ (ACY56284); JOSV, Jos virus IbAn17854 (AED98371); QRFV, Quaranfil virus EGT377 (ACY56282); THOV, Thogoto virus SiAr126 (YP_145794); TLKV, Tyulek virus Leiv152 (AFN73049); UPOV, Upolu virus C5581 (KC506157); and WFBV, Wellfleet Bay virus 10-280-G (KM114305). (B) Amino acid alignment of the matrix (M) proteins of WFBV and Cygnet River virus (CyRV), demonstrating the high identity between the two viruses. Viruses used in the alignment were WFBV 10-280-G (KM114309) and CyRV 10-10646 (AFB81541). (C) Phylogeny based on M amino acid sequences of available quaranjaviruses. The tree is drawn to a scale of amino acid substitutions per site and is midpoint rooted for purposes of clarity only. Viruses are shown as designated or proposed ICTV abbreviation/host/location/strain/year. Viruses (and their GenBank accessions) are as follows: CyRV 10-10646 (AFB81541), QRFV EGT377 (ACY56280), TLKV Leiv152 (AFN73051), and WFBV 10-280-G (KM114309).