HVs [Alpha (α)- and Gamma (γ)-herpesvirinae subfamilies] have been detected in eight families in the suborder Odontoceti (toothed whales): Delphinidae, Iniidae, Kogiidae, Monontidae, Phocoenidae, Physeteridae, Pontoporiidae and Ziphiidae [6,7,8,9,10,11,12,13,14]; and in the Balaenoptera genus (suborder Mysticeti) [15]. α-HVs have been commonly associated with skin lesions and systemic infections [8,9,16,17,18], whereas γ-HVs have been frequently found in genital lesions [7,11,13,19,20,21,22,23,24]. In addition, α- and γ-HV infection of the central nervous system (CNS) has been described in a few cetacean species. More specifically, the characteristic pathological changes of alphaherpesviral encephalitis have been reported in harbour porpoises (Phocoena phocoena) [11,25], in a common bottlenose dolphin (Tursiops truncatus) [26], in a common dolphin (Delphinus delphis) [27], in striped dolphins (Stenella coeruleoalba) [17,27], and in Atlantic spotted dolphins (Stenella frontalis) [27]. However, several cases of α-HV infection in the CNS with histopathological changes not directly attributable to these viruses or in the absence of lesions have also been described [11,17,27,28]. Classical CNS lesions associated, but not exclusively, with the presence of α-HVs consist of meningeal mononuclear cell infiltrates, lymphoplasmacytic perivascular cuffs, microgliosis, intranuclear inclusion bodies (INIBs), and neuronal necrosis and/or associated focal neuronophagic nodules [11,25,26]. Some of these lesions (specifically lymphoplasmacytic perivascular cuffs) have been recently described associated with γ-HV detection in the brain of striped and common bottlenose dolphins [29,30].
A BLAST search (www.ncbi.nlm.nih.gov/blast/Blast.cgi (accessed on 27 December 2021)) was conducted to compare the sequenced products with similar DNA polymerase sequences available from other cetacean HVs described in GenBank. A best fit model was selected by the use of the corrected Akaike information criterion (AICc) and Bayesian information criterion (BIC) for nucleotide substitution for a maximum likelihood (ML) analysis using Mega X [40]. A phylogenetic tree was constructed using the ML statistical method with the Tamura 3-parameter model (T92 + G) for nucleotides [40]. A bootstrap resampling (500 replicates) was used to assess the reliability of the tree.
Nakita Euro Model 12
Classical central nervous system (CNS) lesions associated with the presence of α-HVs consisted of meningeal mononuclear cell infiltrates (Me), lymphoplasmacytic perivascular cuffs (PC), diffuse microgliosis (M), intranuclear inclusion bodies (INIB), Malacia (Ma), neuronal necrosis and associated focal neurophagic nodules (NNs), and haemorrhages (H). OL: other lesions; C-I: co-infections; N.I.: nucleotide identity (with highest query cover); HSV-1: herpes simplex human type 1; α: alphaherpesvirus; γ: gammaherpesvirus; qPCR: quantitative polymerase chain reaction; Ct: threshold cycle; mTª: melting temperature; IHQ: immunohistochemistry; NA: not applicable. Highest nucleotide identities between sequences from our study are indicated in boldface.
Case 2, a subadult male striped dolphin, (194 nt sequence length) presented 4/7 of the evaluated lesions (meningitis, perivascular cuffing, microgliosis, and neuronal necrosis and associated focal neuronophagic nodules). The sequence obtained from this animal showed the highest nt similarity (99.45%) (82% Query Cover (QC)) with a sequence detected in the kidney of a striped dolphin stranded during the CeMV epizootic along the Mediterranean Spanish coast in 2007 (GenBank Acc. No. "type":"entrez-nucleotide","attrs":"text":"GQ888669","term_id":"296840679","term_text":"GQ888669"GQ888669). The animal from our study was also a striped dolphin co-infected with dolphin morbillivirus (DMV) stranded in 2007 in the Canary Islands.
Brain cerebral cortex sample of case 4, a male striped dolphin calf presenting 7/7 of the evaluated brain lesions. (A) Suffusive haemorrhages and malacia are present. Original magnification 10; haematoxylin and eosin staining. Inset: numerous basophilic intranuclear inclusion bodies were observed within the neurons and glial cells (arrowheads). Original magnification 40; haematoxylin and eosin staining. (B) Higher magnification of the basophilic intranuclear inclusion bodies (arrowheads). Original magnification 60; haematoxylin and eosin staining.
Histopathological brain lesions from case 5: (A) Brain cerebral cortex sample of case 5, a juvenile male striped dolphin presenting 7/7 of the evaluated brain lesions. Severe non-suppurative meningitis is present. Original magnification 4; haematoxylin and eosin staining. Inset: Detail of perivascular cuffing and non-suppurative meningitis. Original magnification 10; haematoxylin and eosin staining. (B) Brain cerebral cortex sample of case 5, a juvenile male striped dolphin presenting 7/7 of the evaluated brain lesions. Suffusive haemorrhages, malacia and perivascular cuffing are present. Original magnification 10; haematoxylin and eosin staining. Inset: numerous amphophilic intranuclear inclusion bodies were observed within the neurons and glial cells (arrowheads). Detail of perivascular cuffing (asterisk). Original magnification 40; haematoxylin and eosin staining. (C) Higher magnification of the basophilic intranuclear inclusion bodies (arrowheads). Original magnification 60; haematoxylin and eosin staining.
Case 9, a juvenile female common dolphin (204 nt sequence length), showed the highest nt similarity (99.02% (100% QC) and 99.55 (95% QC)) with sequences detected in tissue pools from common dolphins stranded on the coasts of Portugal in 2011 (GenBank Acc. Nos. "type":"entrez-nucleotide","attrs":"text":"MG437212","term_id":"1529377406","term_text":"MG437212"MG437212 and "type":"entrez-nucleotide","attrs":"text":"MG437210","term_id":"1529377400","term_text":"MG437210"MG437210, respectively). Case 9 displayed 4/7 of the evaluated brain lesions: meningitis, perivascular cuffing, microgliosis and neuronal necrosis and associated focal neuronophagic nodules. This case was also co-infected with DMV.
Case 10, a juvenile female Atlantic spotted dolphin, (189 nt sequence length) showed the highest nt similarity (91.4%-90.21%) (100% QC) with the sequence detected in the brain of case 9 from our study and with sequences detected in common dolphins stranded on the coasts of Portugal in 2011 and 2012 (GenBank Acc. No. MG4337212, "type":"entrez-nucleotide","attrs":"text":"MG437211","term_id":"1529377403","term_text":"MG437211"MG437211 (co-infected with DMV), "type":"entrez-nucleotide","attrs":"text":"MG437210","term_id":"1529377400","term_text":"MG437210"MG437210 and "type":"entrez-nucleotide","attrs":"text":"MG437208","term_id":"1529377394","term_text":"MG437208"MG437208). Case 10 displayed 5/7 of the evaluated brain lesions: meningitis, perivascular cuffing, microgliosis, INIB, neuronal necrosis and associated focal neuronophagic nodules (Figure 4).
Brain cerebral cortex sample of case 10, a juvenile female striped dolphin presenting 5/7 of the evaluated brain lesions. A neuronophagic nodule surrounding a neuron with a basophilic intranuclear inclusion body is present. Original magnification 60; haematoxylin and eosin staining.
Case 11, a calf female Atlantic spotted dolphin (nt sequence of 213 length) showed the highest nt similarity (92.56%) (100% QC) with a sequence detected in a pool of tissue samples from a common dolphin stranded on the coast of Portugal in 2011 (GenBank Acc. No. "type":"entrez-nucleotide","attrs":"text":"MG437213","term_id":"1529377410","term_text":"MG437213"MG437213) and 92.09% nt identity (100% QC) with case 12 from our study. This animal displayed 6/7 of the evaluated brain lesions: meningitis, perivascular cuffing, microgliosis, INIB, malacia, and neuronal necrosis and associated focal neuronophagic nodules (Figure 5). Brucella spp. co-infection was suspected in this animal due to severe lymphohistiocytic meningitis affecting the brain and the spinal cord and confirmed by PCR.
Brain cerebral cortex sample of case 11, a female of Atlantic spotted dolphin calf co-infected with Brucella sp., presenting 6/7 of the evaluated brain lesions. Multiple amphophilic intranuclear inclusion bodies within neurons (arrowheads) and satellitosis are present. Original magnification 60; haematoxylin and eosin staining. Inset: medulla oblongata sample of case No 11. Moderate non-suppurative meningitis is present (asterisk). Original magnification 10; haematoxylin and eosin staining.
Brain cerebral cortex sample of case 12, a juvenile male striped dolphin presenting co-infection with DMV, presenting 7/7 of the evaluated brain lesions. Multiple basophilic intranuclear inclusion bodies within the neurons (arrowheads) can be seen with immunostaining against CDV. Original magnification 60; immunohistochemistry against CDV counterstained with haematoxylin. Inset: Brain cerebral cortex sample of case 12. Details of basophilic intranuclear inclusion bodies within giant syncytial cells (arrowheads) are shown with immunostaining against CDV. Original magnification 60; immunohistochemistry against CDV counterstained with haematoxylin.
Nucleotide phylogenetic tree representing the relationships among cetaceans HVs. The phylogenetic tree was based on the herpesvirus DNA polymerase gene and constructed using the Tamura 3-parameter model (T92 + G) Maximum likelihood (ML), with an estimate of statistical support from 500 bootstrap replicates. Bootstrapping values are indicated as percentages next to the bifurcations (BVs less than 50 were collapsed into a polytomy). The name of each sequence includes the GenBank accession number (when available), cetacean species (B.ph, Balaenoptera physalus; D.le, Delphinapterus leucas; D.de, Delphinus delphis; G.gr, Grampus griseus; K.si, Kogia sima; L.ob, Lagenorhynchus obliquidens; M.de, Mesoplodon densirostris; O.or, Orcinus orca; P.cr, Pseudorca crassidens; P.el, Peponocephala electra; P.ph, Phocoena phocoena; S.ch, Sousa chinensis; S.co, Stenella coeruleoalba; S.fr, Stenella frontalis; S.gu, Sotalia guianensis; T.tr, Tursiops truncatus; and Z.ca, Ziphius cavirostris), the year, the geographic area of the stranding (BeS, Bering Sea; CEAt, Central Eastern Atlantic Ocean; In, Indian Ocean; Me, Mediterranean Sea; NEAt, Northeast Atlantic Ocean; Npa, North Pacific Ocean; NS, North Sea; NWAt, Northwest Atlantic Ocean; Pa, Pacific Ocean; SEAt, South Eastern Atlantic Ocean), and the tissues in which they were detected. Herpesviruses sequences from other species incorporated to the phylogenetic analysis included terrestrial and flying mammals (B.ta, Bos taurus; C.lu.fa, Canis lupus familiaris; C.hi, Capra hircus; C.ca, Cervus canadensis; C.el.ba, Cervus elaphus barbarous; E.fe.ca, Equus ferus caballus; H.sa, Homo sapiens; M.ne, Macaca nemestrina; O.da.st, Ovis dalli stonei; P.fu, Pseudalopex fulvipes; S.ku, Scotophilus Kuhlii; S.sc: Sus scrofa) and reptiles (C.si, Crocodylus siamensis). 2ff7e9595c
Comments