An evolutionary divergent pestivirus lacking the N^pro gene systemically infects a whale species

Figures & data

Figure 1. Phylogenetic reconstruction using maximum likelihood estimation of complete polyprotein of known and putative pestiviruses. Main bootstrap values are presented at nodes. Scale bar indicates number of amino acids changes per site. Taxon names are presented by the virus species and virus abbreviation. Abbreviations: CSFV, Classical swine fever virus; AydinPeV, Aydin-like pestivirus; BDV, Border disease virus; GPeV, Giraffe pestivirus; HoBiPeV, HoBi-like pestivirus; BVDV, Bovine viral diarrhea virus; PAPeV, Proghorn antelope pestivirus; PhoPeV, Phocoena pestivirus; LINDA, Lateral shaking inducing neurodegenerative agent; BuPV, Bungowannah porcine pestivirus; RPeV, Rat pestivirus; BatPeV, Bat pestivirus; APPV, Atypical porcine pestivirus

Figure 2. Genomic characterization of Phocoena pestivirus. (a) Comparison of PhoPeV to other pestivirus genome organization. PhoPeV genome arrangement is similar to other pestiviruses except for the unique absence of N^pro in screened harbour porpoises (n = 7). Alignment of N^pro area of PhoPeV and other pestivirus sequences is zoomed in. Absence of N^pro gene coding sequences is highlighted in grey, conserved area in the capsid (from 221nt) is in bold. C/S cleavage site between N^pro and Capsid is highlighted in pink and indicated with an arrowhead. Virus (Abbreviation – GenBank Accession No.): LINDA pestivirus (LINDAPeV-KY436034), Bungowannah pestivirus (BuPV-EF100713), Classical swine fever (CSFV-X87939), Aydin-like pestivirus (AydinPeV-JX428945), Border disease virus (BDV-AF037405), Giraffe pestivirus (GiPeV-AD144617), Bovine viral diarrhea virus-1 (BVDV-1-M31182), Bovine viral diarrhea virus-2 (BVDV-2-U18059), HoBi-like pestivirus (HoBiPeV-AB871953), Proghorn pestivirus (PAPeV-AY781152). (b) Comparison between PhoPeV sequences in different organs generated from two animals. The lung of animal NS170386 has an insertion (yellow) between p7 and NS2, which sequences originates from a region between NS5A and NS5B (striped yellow square). Duplicated and origin of duplicated sequence are zoomed in and are highlighted in teal colour.

Figure 3. Phocoena pestivirus infects different cell types of harbour porpoises without histopathological changes. PhoPeV RNA expression is visible as bright red cytoplasmic staining in smooth muscle cells in the wall of an intestinal artery, epithelial cells in cortical tubules of the kidney, and neurons in the cerebrum of the brain, based on in situ hybridization (ISH) specific for Phocoena pestivirus (top row). Negative control ISH sections of these stain negative (middle row). Serial sections of these tissues, stained by hematoxylin and eosin, do not show any histopathological changes (bottow row). The narrow clefts in the neuropil of the brain are due to freeze-thaw artifact. Original objective magnifications for all panels: 40×. Artery and kidney were from porpoise NS170386, brain was from porpoise NS170385.

Table 1. Cell types throughout the organ systems of two harbour porpoises (NS170385-86) infected by PhoPeV. Tissue sections were stained for PhoPeV RNA by in situ hybridization.

Organ system	Tissue	Cell types expressing pestivirus RNA
Nervous	Cerebrum	Neuron
	Cerebellum	Neuron
Cardiovascular	Heart ventricle	Cardiomyocyte
	Aorta^a	Smooth muscle cell
	Artery	Smooth muscle cell
Respiratory	Blowhole^a	Keratinocyte
	Trachea	Respiratory epithelial cell, submucosal gland epithelial cell
	Bronchus	Respiratory epithelial cell, submucosal gland epithelial cell, smooth muscle cell
	Lung	Bronchiolar smooth muscle cell, alveolar wall interstitial cell
Digestive	Lip^b	Keratinocyte
	Esophagus	Surface epithelial cell
	Pharynx^a	Surface epithelial cell
	Stomach	Gastric pit epithelial cell, smooth muscle cell
	Intestine	Enterocyte, smooth muscle cell
	Pancreas	Exocrine acinar cell, islet of Langerhans cell
	Liver	Bile duct epithelial cell, Kupffer cell
Urogenital	Kidney	Glomerular cell, tubular epithelial cell, collecting duct epithelial cell, pelvic epithelial cell
	Urinary bladder	Transitional epithelial cell, smooth muscle cell
	Uterus^a	Surface epithelial cell
	Testis^b	Sertoli cell, epididymal duct epithelial cell
Lymphoid	Spleen	Mononuclear cell, smooth muscle cell
	Lung-associated lymph node	Mononuclear cell
	Mesenteric lymph node	Mononuclear cell
Endocrine	Adrenal gland	Cortical cell, medullary cell
	Thyroid gland	Follicle epithelial cell
Integumentary	Skin	Keratinocyte
Musculoskeletal	Skeletal muscle^b	Negative

^aPorpoise NS170386 only.

^bPorpoise NS170385 only.

Table 2. Lesions and levels of PhoPeV RNA (inversely correlated with Ct values) in the tissues of PhoPeV-positive harbour porpoises.

Host_ID	Date of stranding	Days in rehabilitation	Lesions (gross pathology/histology)	Sample material	RT-PCR (Ct)
NS170385*	May 2012	9	COD: bronchopneumonia associated with nematode infection, cerebrum polioencephalitis multifocal mild, hepatitis necrotizing multifocal acute marked. Incidental lesions: adrenalitis with eosinphilic intranuclear inclusions lip ulcus, blowhole ulcer, pyloric stomach focal gastritis	Lung	21.1
				Brain	26.5
				Liver	27.7
				Kidney	15.9
				Spleen	20.2
				Bladder	19.2
				Muscle	19.3
NS170386*	Dec 2008	12	COD: bronchopneumonia associated with parasitic and bacterial infection, marked emaciation. Incidental lesions: epiglottal ulcer, necrotizing pharyngitis, necrotizing ulcerative esophagitis, cholangitis ulcerative dermatitis.	Lung	18.2
				Brain	22.6
				Liver	19.6
				Kidney	16.4
				Spleen	18.8
				Bladder	17.6
				Muscle	21.1
				Skin	25.7
NS190005	Mar 2003	26	COD: bilateral keratoconjunctivitis, pneumonia associated wiht bacterial infection. Incidental lesions: pneumonia associated dermatitis	Kidney	32.5
				Spleen	37.3
				Brain	38.2
NS190017	Apr 2004	35	COD: bronchopneumonia associated wiht lungworm larvae, pneumonia associated with Aspergillus infection. Incidental lesions: ulcura on gential slit, rostral tip of palatum durum and on cornea of left eye, colonic crypt abcesses	Kidney	22.4
				Spleen	25.8
				Brain	24.1
NS190022	Jun 2001	10	COD: pneumonia associated with a bacterial infection. Incidental lesions: oesophageal ulcerations, parasitic infections of stomach and pulmonary artery pneumonia associated lesion lymphadenopathy, pleuritis and pericarditis	Kidney	17.3
				Spleen	19.0
NS190025	Jul 2001	0^#	cerebellar haemorhage focal, oesophageal ulcertion multifocal, conjunctivitis, catarrhal mild, dermatitis	Kidney	37.0
				Spleen	Neg
				Brain	38.0
NS190026	Apr 2001	0^#	advanced state of autolysis, no abnormalities detected	Kidney	34.5
				Spleen	36.7
NS190075	Jan 1998	13 years	COD: pneumonia, hyperplasia of the papilla vater causing obstruction of the pancreatic duct	Kidney	34.0
		8 months^+		Spleen	Neg
				Brain	34.8
NS190100	Sep 2014	0^#	pneumonia and dermatitis	Kidney	20.8
				Spleen	30.7
				Brain	30.8
NS190109	Jul 2005	8	COD: encephalitis associated with herpesvirus infection, bronchopneumonia associated with nematode infection. Incidental lesions: pulmonary arteritis associated with nematode infection	Kidney	17.1
				Spleen	23.0

Notes: COD: cause of death; Ct: cycle threshold.

*Samples were also analyzed by NGS.

^#Dead stranded.

⁺Kept in zoo collection.

Figure 4. Phylogenetic reconstruction using maximum likelihood estimation of partial PhoPeV nucleotide genomes. Two main clades are highlighted as probable genotype 1 and 2. The PhoPeV nucleotide genomes used for analysis were 5′UTR, C, Erns, and E2 (GenBank accession nos. MK910230-37). Bootstrap values are presented at nodes. Scale bar indicates number of nucleotide changes per site LINDA virus was used as outgroup (GenBank accession number KY436034).

Data availability

The sequences generated in this study from two full-length PhoPeV genomes have been deposited under GenBank accession numbers (MK910227-37). Other data are available upon request.