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Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

ABSTRACT: BACKGROUND:Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. RESULTS:A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. CONCLUSION:The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

SUBMITTER: Bercovier H

PROVIDER: S-EPMC1079851 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

Bercovier Herve H Fishman Yolanta Y Nahary Ronen R Sinai Sharon S Zlotkin Amir A Eyngor Marina M Gilad Oren O Eldar Avi A Hedrick Ronald P RP

BMC microbiology 20050317

<h4>Background</h4>Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary.<h4 ...[more]

PMID: 15774009

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Project description:BackgroundKoi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment.ResultsA rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers--two inner primers, two outer primers and two loop primers--was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65 degrees C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV.ConclusionThis paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath.

Project description:Koi herpesvirus (KHV) is highly contagious and lethal to cyprinid fish, causing significant economic losses to the carp aquaculture industry, particularly to koi carp breeders. Vaccines delivered through intramuscular needle injection or gene gun are not suitable for mass vaccination of carp. So, the development of cost-effective oral vaccines that are easily applicable at a farm level is highly desirable. In this study, we utilized chitosan-alginate capsules as an oral delivery system for a live probiotic (Lactobacillus rhamnosus) vaccine, pYG-KHV-ORF81/LR CIQ249, expressing KHV ORF81 protein. The tolerance of the encapsulated recombinant Lactobacillus to various digestive environments and the ability of the probiotic strain to colonize the intestine of carp was tested. The immunogenicity and the protective efficacy of the encapsulated probiotic vaccine was evaluated by determining IgM levels, lymphocyte proliferation, expression of immune-related genes, and viral challenge to vaccinated fish. It was clear that the chitosan-alginate capsules protected the probiotic vaccine effectively against extreme digestive environments, and a significant level (P < 0.01) of antigen-specific IgM with KHV-neutralizing activity was detected, which provided a protection rate of ca. 85% for koi carp against KHV challenge. The strategy of using chitosan-alginate capsules to deliver probiotic vaccines is easily applicable for mass oral vaccination of fish.IMPORTANCE An oral probiotic vaccine, pYG-KHV-ORF81/LR CIQ249, encapsulated by chitosan-alginate capsules as an oral delivery system was developed for koi carp against koi herpesvirus (KHV) infection. This encapsulated probiotic vaccine can be protected from various digestive environments and maintain effectively high viability, showing a good tolerance to digestive environments. This encapsulated probiotic vaccine has a good immunogenicity in koi carp via oral vaccination, and a significant level of antigen-specific IgM was effectively induced after oral vaccination, displaying effective KHV-neutralizing activity. This encapsulated probiotic vaccine can provide effective protection for koi carp against KHV challenge, which is handling-stress free for the fish, cost effective, and suitable for the mass oral vaccination of koi carp at a farm level, suggesting a promising vaccine strategy for fish.

Project description:Long noncoding RNAs (lncRNAs) constitute an emerging group of ncRNAs that modulate gene expression at the transcriptional or translational level. Koi herpesvirus (KHV), also known as Cyprinus herpesvirus type 3 (CyHV-3) and characterized by high pathogenicity and high mortality, has caused substantial economic losses in the common carp and koi carp fisheries industry. In this work, we sequenced the lncRNA and mRNA of host koi carp infected with KHV. A total of 20,178 DEmRNAs were obtained, of which 5021 mRNAs were upregulated and 15,157 mRNAs were downregulated. Both KEGG pathways and GO terms were enriched in many important immune pathways. The KEGG analysis showed that DEGs were significantly enriched in many important immune pathways, such as apoptosis, NOD-like receptor signaling pathway, Jak-STAT signaling pathway, TNF signaling pathway, IL-17 signaling pathway, NF-kappa B signaling pathway, and so on. Furthermore, a total of 32,697 novel lncRNA transcripts were obtained from koi carp immune tissues; 9459 of these genes were differentially expressed. Through antisense, cis-acting, and trans-acting analyses, the target genes of differentially expressed lncRNAs (DElncRNAs) were predicted. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the DElncRNA expression pattern was consistent with the differential mRNA expression pattern. The lncRNA-mRNA network analysis, which included many immune pathways, showed that after KHV infection, the expression of most lncRNAs and their target mRNAs were downregulated, suggesting that these lncRNAs engage in a positive regulatory relationship with their target mRNAs. Considering that many studies have shown that herpesviruses can escape the immune system by negatively regulating these immune pathways, we speculated that these lncRNAs play a significant role in KHV's escape from host immunity. Furthermore, 10 immune-related genes and 20 lncRNAs were subsequently verified through RT-qPCR, to confirm the accuracy of the high-throughput sequencing results. In this study, we aimed to explore lncRNA functions in the immune response of lower vertebrates and provide a theoretical basis for the study of noncoding RNAs in teleosts. Therefore, exploring lncRNA expression in KHV-infected koi carp helped us better understand the biological role played by lncRNA-dependent pathways in aquaculture animal viral infection.

Dataset Information

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

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Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

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