This study identified a novel herpesvirus, OtHV3, in the buffy coats of both wild stranded and healthy California sea lions from a managed collection. While OtHV3 was present in both groups, viral loads identified in the case sea lion that died from B cell lymphoma were magnitudes higher compared to the rest of the study population; the median and range of viral loads in Animal A was 29 009 and 5 205–72 164, while tissue loads from other sea lions ranged from 0.01 to 0.22 viral copies/ng DNA. Interestingly, OtHV3 loads were high in the same organs that were described as having lymphoblastic lymphoma upon histopathology.
Lymphomas have been associated with herpesvirus infection in humans, including HHV4 with Burkitt’s lymphoma and other B-cell lymphoproliferative disorders . While research on viral-associated lymphoproliferative disorders in humans is often limited to epidemiological associations between herpesvirus infections and disease, research with animal models has demonstrated cause and effect. Experimental inoculation of owl monkeys (Aotus sp.) with Saimiriine herpesvirus 2 successfully induced acute lymphocytic leukemia; this outcome was demonstrated using intravenous, subcutaneous, and intradermal inoculations . Similar studies have been reported with marmosets and rabbits [13, 15]. Multiple types of human herpesviruses have been associated with patients with B cell acute lymphoblastic leukemia, especially HHV4 and HHV5 . While the clinical pathology of Animal A was suggestive of acute lymphoblastic leukemia (ALL), the acute versus chronic state of neoplasia in this animal was not examined.
In addition to lymphoma, Animal A had numerous esophageal ulcerative lesions in the distal half to third of the esophagus. Interestingly, the highest viral load found in the case animal was from one of these esophageal ulcers. In humans, the esophagus is the most common nongenital viscera affected by herpesvirus infections, with one study demonstrating that 41 of 50 people with herpes lesions in the esophagus postmortem had no other body part affected . Additionally, neoplastic lymphocytes were clustered in the esophageal mucosa and submucosa which could have caused the ulceration and accounted for the high viral load.
In this study, wild stranded sea lions were more likely to be OtHV3-positive by PCR compared to healthy sea lions from a managed collection. Among the stranded sea lions, those that were yearlings were at the highest risk of OtHV3 infection compared to other age classes. While most of the yearlings in the study had pneumonia, in California sea lions, verminous pneumonia is extremely common in yearling animals due to the high prevalence of the lungworm Parafilaroides decorus; thus the role of a herpesvirus in these cases is hard to determine .
Human herpesviruses, including HHV1, HHV2, HHV4, HHV5, HHV6, and HHV7 have been associated with pneumonia. The prevalence of herpesvirus-associated pneumonia is higher among immunocompromised adults compared to those with competent immune systems, and these pneumonias can be due to endogenous reactivation of latent viral infections or introduction of a new virus . The characterization of pneumonia may be an indicator of herpes infection source; in humans, focal lung infections likely originate from the alveoli, while diffuse interstitial pneumonia may be more likely to be from blood-borne dissemination .
Herpesviruses have been associated with pneumonia, primarily interstitial pneumonia, in terrestrial mammals, including cattle, donkeys, horses, and mice [31–34]. Bovine shipping fever pneumonia is due to co-infections by Mannheimia haemolytica or Pasteurella multocida with a viral infection, including bovine herpesvirus 1 . Similar bacterial-herpesvirus co-infections are found in equine, in which equine herpesvirus type 2 is a predisposing factor for Rhodococcus equi pneumonia in foals . As such, evaluating the potential for bacterial-viral coinfections among yearling sea lions may be an area for future research.
In the current study, it is also plausible that increased viral levels are resultant from rather than causal to concurrent disease. In other mammals, stress is well documented as a cause of herpesvirus reactivation, and different stress factors have been shown to have different effects on herpesviral reactivation [35–39]. The finding of a greater prevalence of OtHV3 in sea lions stranding for reasons other than domoic acid toxicosis may reflect increased stress. There was however, no significant difference in viral loads when comparing the different groups, indicating that reactivation of a latent infection was not present.
Our phylogenetic analyses were largely in agreement with previous herpesviral phylogenies. All herpesviral genera were strongly supported, with the exception of Rhadinovirus. Historically, the Gammaherpesvirinae were divided into two genera, Lymphocryptovirus and the more speciose Rhadinovirus. More recently, Macavirus and Percavirus were recognized as distinct clades and split out from Rhadinovirus, leaving it as somewhat of a catch-all genus . The analyses found strong support for OtHV3 clustering with PhocidHV3, which was identified in Hawaiian monk seals . Although PhocidHV4, from Northern elephant seals (Mirounga angustirostris) , was not included in this analysis, it would have certainly fallen in this cluster due to its extremely close relationship to PhocidHV3 (with 98% amino acid identity over this region, as compared to 71% identity between OtHV3 and PhocidHV3). In the Bayesian but not ML analysis, Phocid HV5 (from harbor seals (Phoca vitulina) ) was also supported as clustering with OtHV3/Phocid HV3, forming a pinniped gammaherpesvirus clade.
It should be noted that the qPCR values are for copies of target DNA detected, which may potentially differ from actual copy numbers and virion present. OtHV3 has not been successfully cultured, and thus, the actual numbers of whole virus in the samples remain unknown. We used dilutions of known copy numbers of OtHV3 DNA as a standard curve. This control is a DNA template and does not reflect loss during extraction. The presence of PCR inhibitors or nucleases may result in falsely low readings. Further, extraction efficiency may differ between tissues and/or other samples, so caution should be used to avoid over-interpretation when comparing different sample types. In this study, a handful of organ tissues from ten sea lions other than Animal A were opportunistically tested for OtHV3 DNA. It is important to note that the qPCR assay was developed for blood, not tissues, and further work is needed to further validate whether or not low levels of OtHV3 were truly present in different organs of different animals.
In conclusion, OtHV3 infected California sea lions may potentially be natural animal models for herpesvirus-associated diseases, including pneumonia and lymphoma. Future studies should further assess potential associations between OtHV3 infections and these diseases in California sea lions.