in 2007 , a 14-year - old male killer whale at a marine park in san antonio , texas , usa , died suddenly without notable premonitory signs . on gross examination , mild multifocal meningeal hyperemia and petechial parenchymal hemorrhage focally extensive tan discoloration and fibrosis were present in the right accessory lung lobe with associated hemorrhage and congestion . tissues fixed in 10% buffered formalin were processed routinely and stained with hematoxylin and eosin for histologic examination . inflammatory lesions of the central nervous system were focused in gray matter of the medulla oblongata , pons , mesencephalan , and cerebellum . blood vessels demonstrated mild to moderate acute necrosis and lymphocytic and contained plasmacytic and neutrophilic infiltrates within vascular walls . encephalitis was characterized by perivascular lymphocytes and fewer plasma cells expanding the virchow - robbins spaces . predominant lesions in the lungs were areas of chronic and active abscessation amid a focally extensive area of mixed inflammation and fibrosis . they were characterized by central ulcerations with necrosis and a mixed inflammatory infiltrate surrounded by variable fibrosis and a rim of epithelial hyperplasia . changes in spleen , lymph node , and kidney included acute edema , congestion , and vascular dilation . conventional diagnostic assays were performed for aerobic , anaerobic , and fungal microbes in liver , lung , kidney , cerebrospinal fluid , and brain . the final diagnosis was fulminant peracute bacteremia and septicemia secondary to a primary viral infection associated with nonsuppurative encephalitis . published etiologic considerations for cetacean nonsuppurative encephalitis include morbillivirus and protozoal infections ( 2 ) . a dna microarray with highly conserved sequences from > 1,000 viruses was selected to screen for known and novel viruses ( 3 ) . total rna was extracted from brain tissue and hybridized to a microarray as described ( 4 ) . analysis of the resulting hybridization pattern demonstrated a strong hybridization signal to many oligonucleotide probes on the microarray from the family flaviviridae , in particular to wnv . consensus reverse transcription pcr primers ( 5 ) targeting wnv were used to confirm the microarray results . hq610502 ) yielded a sequence with 99% nt identity and 100% aa acid identity to wnv strain ok03 ( genbank accession no . eu155484.1 ) , a strain originally identified in oklahoma , usa . to further support a wnv diagnosis , we performed immunohistochemical staining on brain tissue . rockville , md , usa ) with peroxidase - tagged goat antirabbit immunoglobulin g ( dakocytomation , carpinteria , ca , usa ) bridge and 3-amino-9-ethylcarbazole ( dakocytomation ) as the chromogen . this staining demonstrated abundant wnv antigen within the cytoplasm of a small number of neurons and glial cells and in fewer macrophages in the brain tissue ( figure ) . brain specimen from killer whale ( orcinus orca ) with west nile virus infection that died at a marine park , san antonio , texas , usa , 2007 . neurons and glial cells demonstrate abundant intracytoplasmic west nile viras antigen . we evaluated wnv exposure within the same cohort , as well as a geographically distant cohort of whales by using serologic testing . all testing was performed at the same laboratory by using a standard plaque - reduction neutralization test . in this assay , a 90% neutralization cutoff was used ( 6 ) . a 90% plaque - reduction titer serum from the affected whale and 5 cohort killer whales from the same marine park in san antonio as well as 5 whales housed at another facility in orlando , florida , usa , were evaluated . in each facility , the animals have regular contact with each other . the facilities are geographically separated so the animals do not have exposure to those in the other park . all 6 animals from texas had 90% plaque - reduction titers > 10 , ranging from 40 to 80 . these findings broaden the known host tropism of wnv to include cetaceans in addition to previously known pinnipeds . although we can not definitively attribute the cause of death of this whale to wnv , the observed lesions are consistent with those caused by wnv in other animals . the serologic results demonstrate that subclinical infections can occur and that exposure can be variable . both bexar county , texas , and orange county , florida , have had wnv in wildlife since 2002 . mosquito management practices are similar in both facilities and have been expanded since this diagnosis . differences in wnv prevalence or mosquito numbers may have played a role in the different serologic results . health evaluations of free - ranging and captive cetaceans should include wnv serology to assess exposure rates . this report focuses on killer whales , but the  loafing  behavior ( stationary positioning at the water s surface ) is commonly seen in many coastal dolphins , thereby increasing the likelihood of mosquito bites and exposure to wnv . serologic screening of bottlenose dolphins ( tursiops truncatus ) from the indian river lagoon demonstrated wnv titers ( 7 ) . as with many species of birds and mammals , wnv infection carries a risk for zoonotic transmission . until the implications of this infection in marine mammals are better understood , biologists and veterinarians working with cetaceans should consider this possibility . potential viral shedding can occur through the oropharygeal cavity and feces as well as through blood and organs during necropsies . finally , our study demonstrates the broad applicability of using panviral microarray - based diagnostics . even though pcr diagnostics are well developed for wnv , the agent was not initially considered as a potential pathogen in this species . panviral microarray can be used not only to identify novel viruses but also to detect unsuspected agents .
in 2007 , nonsuppurative encephalitis was identified in a killer whale at a texas , usa , marine park . 
 panviral dna microarray of brain tissue suggested west nile virus ( wnv ) ; wnv was confirmed by reverse transcription pcr and sequencing . 
 immunohistochemistry demonstrated wnv antigen within neurons . 
 wnv should be considered in cases of encephalitis in cetaceans .