Sequence Instability in the Proviral Long Terminal Repeat and gag Regions from Peripheral Blood and Tissue-Derived Leukocytes of FIV-Infected Cats during the Late Asymptomatic Phase.

Feline immunodeficiency virus (FIV) infection results in viral persistence, a prolonged asymptomatic phase, and progressive immunopathology. During the asymptomatic phase, a cohort of experimentally FIV-infected cats exhibits features of viral latency in blood suggestive of inactive viral replication. We sought to investigate viral replication activity and genomic stability of the FIV proviral long terminal repeat (LTR) and the 5' aspect of gag over time. FIV-infected cats during the asymptomatic phase demonstrated undetectable plasma FIV gag RNA transcripts and intermittent to undetectable blood-derived cell-associated FIV gag RNA. The LTR sequence demonstrated instability in blood-derived cells over time, in spite of low to undetectable viral replication. Sequence variation in the LTR was identified in CD4+ and CD21+ leukocytes from blood and surgically removed lymph nodes. Three single nucleotide polymorphisms (SNPs) in the LTR were commonly identified. Promoter functionality of a common LTR SNP and rare U3 mutation were examined by reporter gene assays and demonstrated either no change or increased basal FIV promoter function, respectively. In conclusion, this cohort of asymptomatic FIV-infected cats demonstrated instability of the LTR and 5' gag sequences during the study period, in spite of undetectable plasma and rare to undetectable viral gag RNA, which suggests that blood may not accurately represent viral activity in asymptomatic FIV-infected cats.

Portal site metastasis after thoracoscopic resection of a cranial mediastinal mass in a dog.

CASE DESCRIPTION: An 11-year-old castrated male Vizsla was evaluated for excision of a cranial mediastinal mass. CLINICAL FINDINGS: The dog had a 1-month history of a cough that had recently increased in frequency. On physical examination, the dog had a grade 2/6 left systolic heart murmur and multiple subcutaneous masses. A soft tissue mass was observed in the cranioventral aspect of the thorax on radiographs. Results of a CT scan revealed a well-defined, 2.8 × 3.2 × 3.9-cm soft tissue mass in the cranial mediastinum. TREATMENT AND OUTCOME: The dog underwent video-assisted thoracoscopic removal of the mediastinal mass and recovered routinely. Histologic examination of excised tissues revealed malignant thymoma. Approximately 6.5 months after surgery, the dog was evaluated because of polyuria, polydipsia, decreased appetite, and vomiting. On physical examination, masses were found in both axillary regions. Results of serum biochemical analysis indicated hypercalcemia. Thoracic ultrasonography revealed pulmonary metastases and a large mass in the right caudoventral region of the thorax. The dog received supportive care and medical treatment for hypercalcemia, but clinical signs recurred. Euthanasia was elected; necropsy and histologic examination revealed thymic carcinoma. CONCLUSIONS AND CLINICAL RELEVANCE: Descriptions of the development of portal site metastasis in canine patients are rare. In this patient, portal site metastasis developed rapidly after thoracoscopic resection of a malignant thymic mass and was associated with hypercalcemia. As use of thoracoscopic procedures increases in veterinary medicine, it will be important to monitor the development of major complications such as those in the patient of this report.

West Nile virus and non-West Nile virus mortality and coinfection of American crows (Corvus brachyrhynchos) in California.

American crows are acutely sensitive to West Nile virus (WNV) infection, and crow mortality has been used in WNV surveillance to monitor enzootic transmission. However, non-WNV sources of mortality could reduce the reliability of crow death as a surveillance tool. Here, using a combination of histopathologic, toxicologic, virologic, and molecular techniques we describe causes of mortality in 67 American crows (Corvus brachyrhynchos) that were collected from a population in the Sacramento Valley of California in 2012 and 2013. Evidence of infectious disease was detected in 70% (47/67) of carcasses. The majority of deaths were linked to a suite of non-WNV viral, bacterial, and fungal infections (39%; 23/59 cases), WNV (36%; 24/67 cases), and an acute toxic event (25%; 15/59 cases). Coinfections were detected in 20% (12/59) of birds and frequently were associated with WNV and poxviral dermatitis. Inferences about WNV activity based on crow mortality should be supported by laboratory confirmation because crow mortality frequently can be caused by other infectious diseases or toxic events.

Genetic variability and in vitro transcriptional permissibility of primary ovine beta-retrovirus promoter isolates.

OBJECTIVE: To assess genomic sequence conservation and variation in the proviral promoter of enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) in tissue samples from 3 sheep with nasal adenocarcinoma associated with ENTV and 3 sheep with pulmonary adenocarcinoma associated with JSRV and to identify a cell culture system that supports transcriptional activity of the ENTV and JSRV viral promoters. ANIMALS: 6 adult sheep. PROCEDURES: Standard PCR procedures for detection of the ENTV and JSRV long terminal repeat (LTR) promoter region were performed on samples from the 3 nasal adenocarcinomas and 3 pulmonary adenocarcinomas, respectively. The LTRs were cloned into shuttle vectors, amplified, sequenced, and analyzed. The cloned LTR regions were transferred into reporter plasmids and multiple human and ruminant cell lines, and primary cells were transfected with the promoter-reporter plasmids. The viral promoter activity was evaluated by use of an in vitro ß-galactosidase reporter assay. RESULTS: Each isolate had a unique nucleotide sequence. Single nucleotide polymorphisms were the most common LTR mutation and rarely occurred at transcription factor binding sites. Relative to ENTV, the JSRV promoter isolates had a conserved 66-bp U3 insertion, including the lung-specific transcription factor HNF-3ß binding site. Among the cell lines used, human embryonic kidney (293T) and goat synovial membrane cells supported promoter transcription. CONCLUSIONS AND CLINICAL RELEVANCE: The LTRs of ENTV and JSRV have extensive blocks of sequence conservation. Human 293T and goat synovial membrane cell lines may be suitable in vitro cell culture systems for further research of viral promoter functions.