Koi herpesvirus (KHV) has recently been classified as a member of the family of Alloherpesviridae within the order of Herpesvirales. One of the unique features of Herpesviridae is latent infection following a primary infection. However, KHV latency has not been recognized. To determine if latency occurs in clinically normal fish from facilities with a history of KHV infection or exposure, the presence of the KHV genome was investigated in healthy koi by PCR and Southern blotting. KHV DNA, but not infectious virus or mRNAs from lytic infection, was detected in white blood cells from investigated koi. Virus shedding was examined via tissue culture and reverse transcription-PCR (RT-PCR) testing of gill mucus and feces from six koi every other day for 1 month. No infectious virus or KHV DNA was detected in fecal secretion or gill swabs, suggesting that neither acute nor persistent infection was present. To determine if KHV latent infections can be reactivated, six koi were subjected to a temperature stress regime. KHV DNA and infectious virus were detected in both gill and fecal swabs by day 8 following temperature stress. KHV DNA was also detectable in brain, spleen, gills, heart, eye, intestine, kidney, liver, and pancreas in euthanized koi 1 month post-temperature stress. Our study suggests that KHV may become latent in leukocytes and other tissues, that it can be reactivated from latency by temperature stress, and that it may be more widespread in the koi population than previously suspected.

Findings suggest that E macusaniensis may be an important gastrointestinal tract pathogen in camelids of all ages. Clinical signs were frequently nonspecific and were often evident before results of fecal examinations for the parasite were positive. As with other coccidia, severity of disease was probably related to ingested dose, host immunity, and other factors. The clinical and herd relevance of positive fecal examination results must be determined.

Author contributions S.N. and W.G. conceived and designed the overall project. S.S. and C.I.U. assisted with selecting the family, gathering the clinical histories and collecting DNA samples under human subject IRB-approved protocols. S.N., W.G. and I.L. designed the WGS analysis. I.L. performed the WGS analysis and candidate variant filtering. S.N., J.W., A.J.K., J.E.H., A.G.C. and J.H. designed and generated the zebrafish rabl3 mutant lines and performed the cancer studies. J.R.H. and S.N. performed zebrafish histology preparation and analysis. J.D.M. performed and analyzed the AP-MS experiments and CompPASS suite protein interactomics. S.N., W.G. and C.W. conceived and designed the in vitro immunoprecipitation, prenylation assays and HEK293T cell proliferation assays, and P.G., A.B., E.L. and B.U. performed these experiments. S.N. and O.M. designed and performed RASless MEF experiments. J.W.P. performed protein structural modeling. B.C.J. and C.A.F. designed and performed purification of recombinant protein. J.A.P., S.G. and J.D.M. assisted with mass spectrometry analysis. Y.H. assisted with RNA-seq data analysis. M.B.G. performed the zebrafish μCT and bone histomorphometric analysis. O.M., X.W. and J.D.M. provided assistance with tissue culture experiments. C.A.C. and J.A.R. provided analysis of clinical exome sequencing data. C.A.C. and I.L. provided analysis of variants in the Exome Aggregation Consortium.

A high prevalence of vertebral deformities has been observed in various fishes, especially cyprinids, from certain regions of the Willamette River for many years. One proposed source of these deformities is exposure to toxicants. Histological evaluation of affected chiselmouth Acrocheilus alutaceus revealed that all lesions associated with vertebrae were associated with metacercariae of digenean trematodes. Approximately half of the northern pikeminnow Ptychocheilus oregonensis had infections in which metacercariae were associated with these lesions. Metacercariae were also associated with vertebral lesions in three of four affected peamouth Mylocheilus caurinus. Many metacercariae that were present within the vertebral bodies were associated with bony dysplasia and bony proliferation in all three species. We also evaluated the association of the metacercariae with the vertebral deformities, using intact fish that had been cleared with trypsin. Fish from the affected regions had a much higher prevalence of metacercariae and deformities and a greater abundance of metacercariae than those in the reference site. Chiselmouths had more deformities and metacercariae than northern pikeminnow. In all fish species, 77% of deformities were directly associated with metacercariae; in chiselmouths, about 95% of the deformities exhibited this relationship. Two types of metacercariae were identified in affected fish: Apophallus sp. (Heterophyidae) and a neascus type (Strigeidida). The Apophallus sp. appeared to be more closely associated with the skeleton deformities. A Myxobolus sp. morphologically similar to M. cyprini was also associated with the vertebral lesions in about 50% of the northern pikeminnow and 5% of the chiselmouths. Intact plasmodia were found in somatic muscle, and lesions containing free spores were often located at bone surfaces. This survey demonstrates that metacercariae (probably Apophallus sp.) and a Myxobolus sp. are major causes of the vertebral deformities seen in cyprinid fishes from certain regions of the Willamette River.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes devastating epizootics of trout and salmon fry in hatcheries around the world. In laboratory and field studies, epizootic survivors are negative for infectious virus by plaque assay at about 50 days postexposure. Survivors are considered virus free with no sequelae and, thus, are subsequently released into the wild. When adults return to spawn, infectious virus can again be isolated. Two hypotheses have been proposed to account for the source of virus in these adults. One hypothesis contends that virus in the epizootic survivors is cleared and that the adults are reinfected with IHNV from a secondary source during their migration upstream. The second hypothesis contends that IHNV persists in a subclinical or latent form and the virus is reactivated during the stress of spawning. Numerous studies have been carried out to test these hypotheses and, after 20 years, questions still remain regarding the maintenance of IHNV in salmonid fish populations. In the study reported here, IHNV-specific lesions in the hematopoietic tissues of rainbow trout survivors, reared in specific-pathogen-free water, were detected 1 year after the epizootic. The fish did not produce infectious virus. The presence of viral protein detected by immunohistochemistry, in viral RNA by PCR amplification, and in IHNV-truncated particles by immunogold electron microscopy confirmed the presence of IHNV in the survivors and provided the first evidence for subclinical persistence of virus in the tissues of IHNV survivors.