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.

Cyprinid herpesvirus 3 (CyHV-3), commonly known as koi herpesvirus (KHV), is a member of theAlloherpesviridae, and is a recently discovered emerging herpesvirus that is highly pathogenic for koi and common carp. Our previous study demonstrated that CyHV-3 becomes latent in peripheral white blood cells (WBC). In this study, CyHV-3 latency was further investigated in IgM⁺WBC. The presence of the CyHV-3 genome in IgM⁺WBC was about 20-fold greater than in IgM⁻WBC. To determine whether CyHV-3 expressed genes during latency, transcription from all eight open reading frames (ORFs) in the terminal repeat was investigated in IgM⁺WBC from koi with latent CyHV-3 infection. Only a spliced ORF6 transcript was found to be abundantly expressed in IgM⁺WBC from CyHV-3 latently infected koi. The spliced ORF6 transcript was also detectedin vitroduring productive infection as early as 1 day postinfection. The ORF6 transcript fromin vitroinfection begins at −127 bp upstream of the ATG codon and ends +188 bp downstream of the stop codon, +20 bp downstream of the polyadenylation signal. The hypothetical protein of ORF6 contains a consensus sequence with homology to a conserved domain of EBNA-3B and ICP4 from Epstein-Barr virus and herpes simplex virus 1, respectively, both members of theHerpesviridae. This is the first report of latent CyHV-3 in B cells and identification of gene transcription during latency for a member of theAlloherpesviridae.IMPORTANCEThis is the first demonstration that a member of theAlloherpesviridae, cyprinid herpesvirus 3 (CyHV-3), establishes a latent infection in the B cells of its host,Cyprinus carpio. In addition, this is the first report of identification of gene transcription during latency for a member ofHerpesviralesoutsideHerpesviridae. This is also the first report that the hypothetical protein of latent transcript of CyHV-3 contains a consensus sequence with homology to a conserved domain of EBNA-3B from Epstein-Barr virus and ICP4 from herpes simplex virus 1, which are genes important for latency. These strongly suggest that latency is evolutionally conserved across vertebrates.

During neuronal latency of herpes simplex virus (HSV)-1, the latency-associated transcript (LAT) is the only viral gene readily detectable. LAT is required for the high-level reactivation phenotype in animal models. LAT's anti-apoptotic activity was recently demonstrated by our group and it was proposed that LAT's anti-apoptotic function is involved in enhancing the reactivation phenotype. Recently, using chimeric virus CJLAT, it was shown that the reactivation phenotype of LAT− mutant dLAT2903 can be restored to wild-type levels by inserting the bovine herpes virus (BHV)-1 latency-related (LR) gene into the LAT locus of this HSV-1 LAT deletion mutant. Although transcription of the LR gene, like LAT, inhibits apoptosis, LR appears to be multifunctional. To investigate whether the LR gene's anti-apoptotic function was responsible for restoring the high-reactivation phenotype, a mutated BHV-1 LR gene was inserted into the LAT locus of HSV-1 generating the chimeric virus CJLATmut. This mutation consists of three stop codons inserted just after the ATG of the first LR open reading frame (ORF2). In plasmids and in a BHV-1 mutant, this mutation eliminated the LR gene's anti-apoptotic activity, strongly suggesting that ORF2 encodes a protein responsible for LR's anti-apoptotic activity. Reactivation of the CJLATmut virus, in both rabbits and mice, was significantly lower than in wild-type McKrae virus (P=0·0001 and P=0·0003, respectively) and CJLAT virus, containing wild-type LR in place of LAT (P<0·0001) and was similar to LAT− dLAT2903 (P=0·8 and P=0·7, respectively). Thus, disruption of BHV-1 LR ORF2 eliminated the high-reactivation phenotype.