Investigation of koi herpesvirus latency in koi.

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.

Results of total DNA measurement in koi tissue by Koi Herpes Virus real-time PCR.

Koi Herpes Virus (KHV) has been classified recently as a member of the Alloherpesviridae within the Herpesvirales order (Waltzek et al., 2005). Although one of the unique features of Herpesviridae, the sister family of Herpesvirales, is latent infection, it has not been demonstrated consistently that KHV of Alloherpesviridae can cause latent infection and be reactivated from latency. To investigate if KHV genomic DNA is present in koi exposed to KHV infection, 10 healthy fish were investigated from a koi population with a history of a KHV outbreak. No gross lesions or microscopic changes were observed at necropsy or by histological examination. No infectious virus was isolated from either the blood plasma or tissues. However, KHV DNA was detected in the white blood cells of nine of the ten fish by real-time PCR and PCR-Southern blot. KHV DNA was also detected in the brain, eye, spleen, gills hematopoietic kidney, trunk kidney, and intestine of nine of the ten fish by PCR-Southern blot. Interestingly, KHV DNA was also detected in the intestinal contents from seven of ten koi. Portions of major capsid gene DNA, amplified from two of the ten koi WBCs, were found to be identical to KHV-U. This study demonstrated that KHV genomic DNA can be detected in normal koi exposed previously to KHV and suggests that KHV becomes latent in fish.

Reduction of herpes simplex virus type-2 replication in cell cultures and in rodent models with peptide-conjugated morpholino oligomers.

BACKGROUND: Genital herpes, caused by herpes simplex virus type-2 (HSV-2), is a recurrent, lifelong disease affecting tens of millions of people in the USA alone. HSV-2 can be treated therapeutically with acyclovir (ACV) and its derivatives; however, no treatment can prevent HSV reactivation. Novel topical anti-HSV microbicides are much needed to reduce HSV-2 transmission and to treat primary or reactivated infections, especially for ACV-resistant strains. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are single-stranded DNA analogues that enter cells readily and can reduce target gene expression through steric blockage of complementary messenger RNA (mRNA). METHODS: We investigated the antiviral activities of PPMOs targeted to the translation start-site regions of the mRNA for two HSV-2 immediate early genes, immediate early protein (ICP)0 and ICP27, and two early genes, unique long gene (UL)30 and UL39. RESULTS: In cell cultures, PPMOs targeting ICP0 or ICP27 mRNA were found to be highly effective against two strains of HSV-2, one of which was ACV-resistant. In vivo, daily topical applications of up to 1 mM ICP27 PPMO caused no gross or microscopic damage to the genital tract of uninfected BALB/c mice or cotton rats. Cotton rats receiving topical application of ICP27 PPMO 24 h after HSV-2 inoculation showed a reduction in genital lesions and a 37.5% reduction in mortality at 14 days post-infection. Mice receiving topical application of 100 µM of an ICP27 and ICP0 PPMO combination before HSV-2 inoculation had no detectable viral replication in the genital tract at 3-5 days post-infection. CONCLUSIONS: These results demonstrate that topically applied PPMOs hold promise as candidate antiviral microbicides against HSV-2 genital infection.

Inhibition of HSV-1 ocular infection with morpholino oligomers targeting ICP0 and ICP27.

Alternative therapies are needed for HSV-1 infections in patients refractory to treatment with Acyclovir (ACV) and its derivatives. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA analogues that enter cells readily and reduce target gene expression through steric blockage of complementary RNA. When applied before or soon after infection PPMO targeting the translation-start-site regions of HSV-1 ICP0 or ICP27 mRNA reduced HSV-1 plaque formation by 70-98% in vitro. The ICP0 PPMO also reduced ACV-resistant HSV-1 (strain 615.9) plaque formation by 70-90%, while an equivalent dose of ACV produced only 40-50% inhibition when the treatment was applied between 1 and 3hpi. Seven daily topical treatments of 100microg ICP0 PPMO caused no gross or microscopic damage to the corneas of uninfected mice. Topical application of 10microg ICP0 PPMO to the eyes of HSV-1 infected mice reduced the incidence of eye disease by 37.5-50% compared to controls. This study demonstrates that topically applied PPMO holds promise as an antiviral drug candidate against HSV-1 ocular infection.

Characterization of Cervidpoxvirus isolates from Oregon, California, and eastern Canada.

The present report describes the analysis of 4 Deerpox virus isolates from California, Oregon, and Ontario, Canada. All 4 isolates were associated with cutaneous crusting lesions. Examination of selected samples by electron microscopy demonstrated that the viruses were morphologically similar to orthopoxviruses. Phylogenetic analysis of the A21 gene, which is found in all poxviruses, indicated that the 4 isolates form a lineage distinct from other members except for those belonging to the genus Cervidpoxvirus of the subfamily Chordopoxvirinae. Members of the Cervidpoxvirus lineage encode a set of genes not found in other poxviruses. These include homologs of genes encoding interleukin 1 receptor antagonists (IL-1Ra) and C-type lectin-like receptors (CTLR). In the current investigation, genes encoding homologs of IL-1Ra and CTLR were amplified from all the isolates and were found to be closely related to orthologs found in the Cervidpoxvirus genus, which further supports the inclusion of these isolates in the Cervidpoxvirus genus.

Strong population structure characterizes weediness gene evolution in the invasive grass species Brachypodium distachyon.

Mediterranean annual grasses have invaded California and have replaced vast areas of native grassland. One of these invasive grasses is Brachypodium distachyon, a new model species for the grasses with extensive genomic resources and a nearly completed genome sequence. This study shows that the level of genetic variation in invaded California grasslands is lower compared to the native range in Eurasia. The invaded regions are characterized by highly differentiated populations of B. distachyon isolated by distance, most likely as a result of founder effects and a dearth of outcrossing events. EXP6 and EXP10 encoding alpha-expansins responsible for rapid growth, and AGL11 and AGL13 encoding proteins involved in vegetative phase regulation, appear to be under purifying selection with no evidence for local adaptation. Our data show that B. distachyon has diverged only recently from related Brachypodium species and that tetraploidization might have been as recent as a few thousand years ago. Observed low genetic variation in EXP10 and AGL13 appears to have been present in Eurasia before tetraploidization, potentially as a result of strong selective pressures on advantageous mutations, which are most likely responsible for its fast growth and rapid completion of its life cycle.