Three New Orbivirus Species Isolated from Farmed White-Tailed Deer (Odocoileus virginianus) in the United States

Abstract

We report the detection and gene coding sequences of three novel Orbivirus species found in six dead farmed white-tailed deer in the United States. Phylogenetic analyses indicate that the new orbiviruses are genetically closely related to the Guangxi, Mobuck, Peruvian horse sickness, and Yunnan orbiviruses, which are thought to be solely borne by mosquitos. However, four of the six viruses analyzed in this work were found as co-infecting agents along with a known cervid pathogen, epizootic hemorrhagic disease virus-2 (EHDV-2), raising questions as to whether the new viruses are primary pathogens or secondary pathogens that exacerbate EHDV-2 infections. Moreover, EHDV-2 is known to be a Culicoides-borne virus, raising additional questions as to whether Culicoides species can also serve as vectors for the novel orbiviruses, if mosquitoes can vector EHDV-2, or whether the deer were infected through separate bites by the insects. Our findings expand knowledge of the possible viral pathogens of deer in the United States. Moreover, due to the close genetic relatedness of the three new orbiviruses to viruses that are primary pathogens of cattle and horses, our findings also underscore a crucial need for additional research on the potential role of the three new orbiviruses as pathogens of other animals.

Keywords: Odocoileus virginianus; deer farming; orbivirus; reovirus; white-tailed deer.

Figure 1

Geographic information regarding white-tailed deer infection sites. (A) Map location of the two states (Florida and Pennsylvania) wherein the dead deer of this study originated. (B) Location of counties in Florida that contained the deer farms of this report. (C) Location of county in Pennsylvania that contained a deer farm in this report.

Figure 2

Virus-induced cytopathic effects (CPEs) in C6/36 cells. Panels (A–C): Mock-infected cells 3, 5, and 9 days post-inoculation (dpi) with phosphate-buffered saline (PBS). Cytopathic effects induced by the virus(es) stemming from spleen homogenates are shown for (D) OV610 at 9 dpi, (E) OV617 at 8 dpi, (F) OV682 at 9 dpi, (G) OV862 at 5 dpi, (H) OV867 at 4 dpi, and (I) OV926 at 9 dpi. All images were taken at an original magnification of 400×.

Figure 3

Ultrastructure of orbivirus OV682 in C6/36 cells. (A) Fragment of an infected cell with virus inclusion (“virus factory”) containing maturing virus particles (thick arrows) and virus-containing vacuoles (big arrowheads). Small arrowheads point out virus particles at the periphery of the cytosol. The letter N identifies a fragment of the host cell nucleus. Scale bar = 1 um. (B) Fragment of a cell demonstrating a portion of a virus factory with maturing particles. The letter N identifies a fragment of the host cell nucleus. Scale bar = 1 nm. (C) Fragment of an intracytoplasmic virus-containing vacuole. Scale bar = 1 nm.

Figure 4

Maximum Likelihood (ML) phylogram of orbivirus T2 proteins. Shown is a ML phylogram depicting the relationship of the six CHeRI orbiviruses of this work to representatives of the genus Orbivirus based on the amino acid sequences of the T2 proteins coded by their innermost subcore capsid VP3 genes. Bootstrap values are given at each node and the branch lengths represent the number of inferred substitutions as indicated by the scale.

Figure 5

Sequence identity matrix of orbivirus T2 proteins. Shown is a sequence identity matrix depicting the amino acid percent identity of the six CHeRI orbiviruses of this work to 28 other orbiviruses based on the deduced amino acid sequences of the T2 proteins coded by their innermost subcore capsid VP3 genes.

Figure 6

Maximum Likelihood (ML) phylogram of orbivirus RdRp proteins. Shown is a ML phylogram depicting the relationship of the six CHeRI orbiviruses of this work to representatives of the genus Orbivirus based on based on the deduced amino acid sequences of the RdRp proteins coded by their VP1 genes. Bootstrap values are given at each node and the branch lengths represent the number of inferred substitutions as indicated by the scale.

Figure 7

Sequence identity matrix of orbivirus RdRp proteins. Shown is a sequence identity matrix depicting the amino acid identity of the six CHeRI orbiviruses of this work to 28 other orbiviruses based on the deduced amino acid sequences of the RdRp proteins coded by their VP1 genes.

Figure 8

Maximum Likelihood (ML) phylogram of orbivirus major outer capsid proteins. Shown is a ML phylogram depicting the relationship of the six CHeRI orbiviruses of this work to representatives of the genus Orbivirus based on the deduced aa sequences of the major outer capsid proteins coded by their VP2 genes. Bootstrap values are given at each node and the branch lengths represent the number of inferred substitutions as indicated by the scale bar.

Figure 9

Sequence identity matrix of orbivirus major outer capsid proteins. Shown is a sequence identity matrix depicting the amino acid percentage identity of the six CHeRI orbiviruses of this work to 28 other orbiviruses based on the deduced amino acid sequences of the major outer capsid proteins coded by their VP2 genes.