Isolation And Identification Of A Koi Herpesvirus And Development Of Method For Detection Of Koi Herpesvirus In Lytic And Latent Infection

Koi herpesvirus(KHV),was renamed as Cyprinid herpesvirus 3(CyHV-3)in 2005,is a member of the Alloherpesviridae.KHV is a pathogenic of common carp(Cyprinus carpio)and koi with mortality rate from 80 to 100%.Since the first report in 1998,many outbreaks of KHV disease were reported in the worldwide expect North Africa.Because of its worldwide spread and the economic losses it caused,KHV became rapidly a notifiable disease and an object of global import and export trade's testing.KHV has strong pathogenicity,high incidence,high mortality and rapid spread,which pose a threat to carp culture farms and has caused severe economic losses in common and koi carp culture industries.The infection of KHV can be divided into two phase,lytic infection and latent infection.During lytic infection,lots of KHV virus produced and cause sever clinic symptom.During latent infection,viruses stay in the nucleus of the host cell with a non-integrated state.Under certain conditions,the virus will be re-activated to cause clinical symptoms and death.Latent infection is one of the major reasons for this disease worldwide spread.Therefore,it is important to establish a rapid and accurate method for the detection of KHV in the prevention and control of this disease.In this study,a KHV strain was isolated from an outbreak infectious disease of common carp in Sichuan,and a phylogenetic tree was constructed by using TK gene.According to the manifestations of lytic infection and latent infection of KHV,the rapid detection assays were estabilished in lytic and latant infection,respectively.And then the methods were used for testing clinical samples.The result demonstrated that the methods established in this study are highly sensitivity and specificity.This study provides an experimental basis for epidemiological investigation and prevention of KHVD.This study covers three chapters as below.1.Isolation and identification of a Koi Herpesvirus and phylogenetic analysis from Sichuan ProvinceIn May 2015,an unknown disease caused severe economic losses in a Common carp(Cyprinus carpio)culture pond in Sichuan Province.The mortality rate reaches up 80%.Necropsy,bacteriologic test,histological examination,pathogenicity tests,virus isolation,PCR assay and phylogenetic analysis were performed to explore its causes and regularty of epidemic.Principal symptoms included sunken eyes,hemorrhage at the base of the pectoral and pelvic fins,severe gill necrosis and swollen kidney.Bacteriologic test was negative.Histopathologically,the most important changes were observed in the gills and kidneys.The respiratory of gill lamellae was swelling and/or dropping and congestion of the blood vessels in the gill filaments.The base of branchial epithelium also exhibit hyperplasia,hypertrophy and cellular layer increased.The obvious disorder in structure,swelling,and disintegration and necrosis were found in epithelial cells of renal tubulus.Positive results were obtained from all detected samples according to a standard PCR diagnosis of KHV Sph gene proposed by Office International des Epizooties(OIE)by using gills and kidney tissue DNA isolated from diseased Common carp.After filtration treatment,the kidney and liver homogenate was injected intraperitoneally into 20 healthy common carp,and the injected carp showed similar clinical symptoms as the fish that was naturally infected.Common Carp died acutely in the trial group with a cumulative mortality rate 90%.Tissue suspension was inoculated to the Common Carp Brain(CCB)monolayer cells and a typical cytopathic effect(CPE)was observed in CCB cells after three blind passages.Icosahedral viral particles were observed in cells under the electron microscope.The virus particles were spherical in shape measuring 180?200 nm in diameter.Based on neighbor-joining analyses of the TK gene sequences,phylogenetic tree was constructed and the result showed that the isolate belongs to Asian 1 genotype.This is the first report of KHV infection in cultured common carp associated with high mortality in Southwestern China.The result present in this study is important for virus evolution,classification,viral diagnosis and disease control.2.Development of a method for detection of Koi herpesvirus in lytic infectionTo develop a rapid,accurate and suitable clinical application assay for detect Koi herpesvirus(KHV)in lytic infection.Loop-mediated isothermal amplification(LAMP)was selected for this study.Four primers were designed for LAMP and two primers designed by oligo software to improve the cycle rate of LAMP reaction.KHV Sph was selected as the target gene.After optimization of the reaction conditions,sensitivity and specificity of the assay were analyzed.The method established in this study is highly specific,no cross-reactions with CyHV-1?CyHV-2 and AngHV.The method is highly sensitivity with a limit detection of 13.9 copies/?L.The clinical samples were collected from five carp and Koi culture pond of Sichuan province by non-lethal sampling(gill swab).The positive rate was 31.40%(27/86),which it is the same as the result of PCR.In this study,a high sensitivity,high specificity,rapid and simple method was estabilished for detection KHV in lytic infection.The method established in this study is suitable for rapid diagnosis of KHVD in wild.3.Development of assays for detection of Koi herpesvirus in latent infectionTo establish a rapid,sensitive and specific assay for the detection of KHV in latent infection,Real-time PCR was developed with specific primers designed to target the special sequence of KHV Sph gene.By optimization of reacting conditions,and the specificity and sensitivity of the method were evaluated and the preliminary application of clinical samples was studied.The results showed that the assay were capable of KHV latent detection without cross-reaction with other fish pathogenic viruses,including Carp pox herpesvirus(CyHV-1)?Goldfish Herpes Virus(CyHV-2)and eel herpes virus(AngHV-1).The limited detection concentration of Real-time PCR was 42 copies/?L.Moreover,63 clinical samples were detected by Real-time PCR and LAMP.The result showed that 18 samples come from the fish which have been confirmed has KHV disease history,18 out of 18 samples were positive,the positive rate was 100%(18/18);15 samples come from the fish which present KHV clinical sign and confirmed by PCR,15 out of 15 samples were positive,the positive rate was 100%(15/15),30 samples come from the fish which has no any clinical symptoms,6 out of 30 samples were positive,the positive rate was 20%(6/30).In conclusion,the development of this assay provides a rapid,sensitive and reliable molecular tool for detection of KHV latent infection and supply technical support for molecular epidemiology investigation and prevention of KHVD.