Diagnostic sensitivity and specificity of in situ hybridization and immunohistochemistry for Eastern equine encephalitis virus and West Nile virus in formalin-fixed, paraffin-embedded brain tissue of horses.

Immunohistochemistry (IHC) and in situ hybridization (ISH) can be used either to detect or to differentiate between Eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) within formalin-fixed, paraffin-embedded (FFPE) brain tissue of horses. To compare the diagnostic sensitivity and specificity of ISH and IHC, FFPE brain tissue from 20 EEEV-positive horses and 16 WNV-positive horses were tested with both EEEV and WNV oligoprobes and EEEV- and WNV-specific antibodies. Reverse transcription polymerase chain reaction (RT-PCR) for detection of EEEV and WNV was used as the gold standard to confirm infection. All horses that tested positive for EEEV by RT-PCR also tested positive by IHC and ISH, except for 1 case that was false-negative by ISH. In contrast, all horses that tested positive for WNV by RT-PCR tested negative by IHC and only 2 horses tested positive by ISH. No false-positives were detected with either method for both viruses. Both IHC and ISH are highly specific and sensitive diagnostic methods to detect EEEV in equine FFPE brain tissues, although neither appear effective for the diagnosis of WNV in equine neurologic cases.

Novel iridovirus in a nautilus (Nautilus spp.).

Intracytoplasmic inclusion bodies suggestive of iridovirus infection were observed in formalin-fixed, paraffin-embedded tissues from a nautilus (Nautilus spp.) that died without premonitory signs. Transmission electron microscopy revealed enveloped, hexagonal, viral particles that measured approximately 176 nm in diameter. Virions contained a dense central core and morphology typical of iridoviruses. Extracted DNA was amplified using primers homologous to conserved iridovirus sequences. The amplicons were cloned, sequenced, and determined to be approximately 60% similar to reported amphibian iridovirus sequences. A polymerase chain reaction-generated digoxigenin probe was used to detect viral nucleic acid in tissue sections by DNA in situ hybridization and high-affinity cytochemistry. The detected nucleic acid corresponded to the inclusion bodies observed microscopically. This represents a novel iridovirus of mollusks.

Persistent viral shedding during asymptomatic Aleutian mink disease parvoviral infection in a ferret.

A 2-year-old domestic ferret that appeared clinically healthy was repeatedly seropositive for Aleutian mink disease parvovirus (ADV) over a 2-year observation period. Antibody titers, determined by counter-immunoelectrophoresis, ranged from 1024 to 4096. Viral DNA also was identified in serum, urine, feces, and blood cell fractions by polymerase chain reaction analysis. Ultimately, DNA in situ hybridization revealed ADV DNA in histologic sections of various tissues and organs. These data indicate that this asymptomatic ferret was persistently infected with ADV.

Molecular diagnosis of paramyxovirus infection in snakes using reverse transcriptase-polymerase chain reaction and complementary deoxyribonucleic acid:ribonucleic acid in situ hybridization.

Identification of ophidian paramyxovirus (OPMV) nucleic acid was accomplished in 11 of 14 snakes by a reverse transcriptase-polymerase chain reaction (RT-PCR) assay that detected a 153-bp region of the OPMV genome in total RNA extracted from paraffin-embedded tissues and cell culture. The RT-PCR protocol amplified a portion of the OPMV RNA genome, producing a 153-bp complementary DNA (cDNA) product from both fresh and paraffin-embedded tissue samples. In addition, cDNA:RNA in situ hybridization localized OPMV in formalin-fixed, paraffin-embedded tissue specimens to specific tissues and cells. This latter technique increased the degree of specificity with which a diagnosis of OPMV could be made.