Investigation of koi herpesvirus latency in koi

Abstract

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.

Fig. 1.

Real-time PCR and PCR with Southern blotting of KHV PCR amplicons from koi WBC or plasma. (A) Standard curve using threshold cycle values to calculate the analytic sensitivity of KHV genome equivalents with the real-time TaqMan PCR. The assay detects a range from 10 to 10⁸ copies of a plasmid bearing the KHV target sequences. (B) KHV DNA copy number per microgram of total WBC DNA isolated from six koi (designated K1 to K6) that were bled three times (at 2 weeks, 1 month, and 2 months following arrival at OSU-SDL). (C and D) Autoradiogram of the KHV DNA polymerase gene. Amplicons were amplified with primers KHVDF-242 and KHVDR-242 from six koi on three occasions (2 weeks, 1 month, and 2 months following arrival at OSU-SDL) after testing for the presence of KHV DNA in WBC (C) and plasma (D). The positive control (P) was KHV-U DNA. N, negative control.

Fig. 2.

Detection of the KHV mRNA of DNA polymerase gene and major capsid gene from WBC total RNA. (A) RT-PCR of WBC total RNA with primers KHVDF-242 and KHVDR-242 (Table 1). (B) RT-PCR of WBC total RNA with primers KHVC382F and KHVC382R specific for a portion of major capsid gene at 382 bp. (C) RT-PCR of 18S RNA using Quantum RNA 18S internal standards (Ambion) with an 18S PCR primer pair (the expected product size is 324 bp). Lane 1, KHV-U DNA; lane 2, WBC total DNA of four koi latently infected with KHV; lanes 3 and 4, total RNA of KF-1 cells infected with KHV at 8 days postinfection; lanes 5 and 6, WBC total RNA of four koi latently infected with KHV. Lanes 3 and 5 are without reverse transcriptase, and lanes 4 and 6 are with reverse transcriptase in the RT reaction.

Fig. 3.

Water temperature change and detection of KHV DNA in fecal and gill swabs. (A) Tank water temperature change. Vent and gill swabs were collected from all six koi every other day starting at day 2 post-temperature increase until day 16 post-temperature increase. (B) Percentage of KHV swabs testing positive by real-time PCR for KHV DNA.

Fig. 4.

Examination of KHV DNA in tissue from koi that died during the reactivation study. (A) KHV DNA copy number per microgram of total DNA isolated from two dead koi, K5 and K6, that died during temperature stress. (B) Comparison of the average KHV DNA copy number per microgram of total DNA between tissues of K5 and K6, which died during temperature stress, and tissues of K1 to K4 collected after stress. ***, P < 0.001, in comparison with the average of KHV DNA from K5 and K6.

Fig. 5.

Phylogenetic analysis of the PCR DNA sequence of the KHV DNA polymerase gene. KHV DNA recovered from tested koi was compared to the KHV U.S. strain (DQ657948), KHV Israeli strain (DQ177346), KHV Japanese strain (AP008984), and a recent isolate (DQ128163) from common carp in the United States. The scale bar represents the genetic distance (nucleotide substitutions per site). K1 and K3, KHV DNA amplified from WBC from koi with a history of KHV infection; K5 and K6, KHV DNA amplified from tissue collected during necropsy of fish that died during temperature stress; KHVI and KIII, KHV DNA amplified from WBC total DNA from koi with no known KHV exposure; Kmix, KHV DNA amplified from combined WBC total DNA of four koi obtained from a local pet store.

Fig. 6.

KHV DNA distribution in tissue of previously exposed koi (K1 to K4). (A) Real-time PCR of the KHV DNA from koi tissue after temperature stress. (B) PCR followed by Southern blotting of the KHV PCR amplicons from koi tissues collected after temperature stress. Shown is an autoradiograph of amplicons with specific primers targeting the KHV ORF26 DNA sequence (KHVDF-447 and KHVDR-447) and probed with the 263-bp DNA probe which is internal to the amplification region by primers KHVDF-447 and KHVDR-447. Template DNA was as follows: lane 1, brain; lane 2, spleen; lane 3, hematopoietic kidney; lane 4, gills; lane 5, heart; lane 6, eye; lane 7, intestine; lane 8, liver; lane 9, trunk kidney; lane 10, intestinal content; lane 11, gonad; lane 12, pancreas; P, positive control (KHV-U viral DNA); N, negative control.

Fig. 7.

Detection of the KHV PCR amplicons from WBC total DNA from koi with no known history of KHV exposure. (A) Real-time PCR of KHV DNA from WBC collected at 2 weeks following arrival. The y axis represents the KHV DNA copy number per microgram of each koi WBC isolated from 1 ml of blood. (B) Autoradiogram of KHV PCR amplicons hybridized with KHV DNA probe which is internal to the PCR primer amplification region shown in Fig. 1. Lanes I to V, PCR amplicons of WBC total DNA isolated from KI to KV; P, positive control (KHV-U DNA); N, negative control.

Fig. 8.

Detection of the KHV PCR amplicons from tissue total DNA from koi with no known history of KHV exposure. (A) Real-time PCR of the KHV DNA from koi tissue of KI to KV. x axis, total tissue DNA isolated from 50 to 100 μg of tissue; y axis, KHV DNA copy number per microgram of total DNA from each tissue. (B) Comparison of the average KHV DNA copy number per microgram of total DNA between tissues of koi K5 and K6, which died during temperature stress, and tissues of KI to KV collected 2 months later. ***, P < 0.001, in comparison with the average of KHV DNA from K5 and K6. (C) PCR followed by Southern blotting of the KHV DNA of tissue from koi KI to KV. Shown is an autoradiogram of KHV DNA polymerase gene amplicons, using primers KHVDF-242 and KHVDR-242, hybridized with the KHV DNA probe, which is internal to the PCR primer amplification region as shown in Fig. 1. The positive control (P) is KHV-U DNA. Template DNA was as follows: lane 1, brain; lane 2, spleen; lane 3, hematopoietic kidney; lane 4, gills; lane 5, heart; lane 6, eye; lane 7, intestine; lane 8, liver; lane 9, trunk kidney; lane 10, fecal; lane 11, gonad; lane 12, pancreas; P, positive control; N, negative control.

Fig. 9.

KHV antibody ELISA. Results of serum collected at three occasions (2 weeks, 1 month, and 2 months following arrival at OSU-SDL). An optical density (OD) below 0.24 is considered negative. The six koi, K1 to K6, were from populations with a history of KHV infection. The asterisk indicates that the koi blood is positive for the KHV antibody. +, positive control; −, negative control.