Application Of Molecular Biology To Rapid Detection Of Equine Rhinopneumonitis Virus

1. A multiplex PCR method was established to simultaneously identify and distinguishequine herpesviruses EHV-1, EHV-2 and EHV-4 in the same reaction within several hours,using 3 pairs of type-specific primers chosen from the glycoprotein B (gB) coding region ofeach serotype. The PCR amplicons of EHV-1, EHV-2 and EHV-4, as predicted 226, 333,570 base pairs, respectively, could be readily separated by gel electrophoresis. Whencompared to the absence of speific DNA bands in herpesviruses (BHV-1,PHV-1,GHV-1,AHV-1) from other animals, the specificity of these amplifications was confirmed bydetermining their nucleotide sequences of the PCR products, consisting with the publishedsequence of these gB genes. And the assay was sensitive, indicating a detection limit of 103TCID₅₀. Tissues from one seropositive horse of post-arrival quarantine was positive forEHV-1 by PCR whereas some nasaopharyngeal swabs from seropositive horses ofpre-export quarantine were positive for EHV-4. Infectious viruses were not recovered inany tested specimens from which the animals did not show any clinical signal of EHV-1 orEHV-4 infections. It is showed that multiplex PCR assay, which is quick, specific, sensitive,and reliable, seems to be a practical alternative to identify the low level of non-infectiousequine herpesviruses or the presence of latent forms in horse tissue.2. To enhance the sensitivity of inspection for equine herpesviruses, a novel Eva Green dyewas applied in fluorescence PCR to detect and distinguish 4 types of Equine Herpesviruses(EHV-1, EHV-2, EHV-3 and EHV-4) by specific primers. This assay was more sensitive inthat it indicated a detection limit of 1 fg of viral DNA (16 molecules of EHV). And itsspecificity was confirmed by the presence of dissociation curves of the PCR products ofEHV-1, EHV-2, EHV-3 and EHV-4 at 79℃, 86℃, 84℃and 81℃, respectively. Tissuesamples from one seropositive horse of post-arrival quarantine was positive for EHV-1 byfluorescence PCR but negative by virus isolation, whereas fifteen nasaopharyngeal swabsfrom seropositive horses of pre-export quarantine were positive for EHV-4 by fluorescencePCR but negative by virus isolation. EHV-4 DNA detected by fluorescence PCR was lowand of short duration in Peripheral blood leucocytes (PBLs), but high and of long duration in nasopharyngeal secretions (NS) after experimental challenge. It is showed thatfluorescence PCR assay, which is quick, specific, sensitive, and reliable, seems to be apractical alternative to identify the low level of non-infectious equine herpesviruses or thepresence of latent forms in horse tissue.3. To identify various equine pathogens, the highly conservative DNAs of Equineherpesvirus 1, Equine arteritis virus, Equine influenza virus, Equine infectious anaemiavirus and Eastern equine encephalomyelitis virus were acquired by molecular cloning, andthen spotted on the treated glass slides as the diagnostic gene-chip. And the cDNAsreverse-transcripted from RNAs of samples were labeled with Cy5 fluorescence asprobes. Following specific hybridization of deposited gene chip and labeled probes,fluorescence signals were scanned by laser scanner and the obtained imagewas analyzed by QiamtArray software with the digital computer. The resultsshowed that the prepared gene chip could detect and distinguish the five equine viruses.And its sensitivity was about 25 copies of viral genomes. The hybridization specificity wasconfirmed by the presence of red fluorescence signals on the corresponding sites withsamples from the five relevant viruses in horses and by the absence of positive signals withthe specimens from irrelevant viruses in other animals. Peripheral blood leucocyte (PBL)from some seropositive horses of post-arrival quarantine was negative by virus isolation butpositive for EHV-1 and EAV by gene chip. The evidence suggests that gene chip, which isquick, specific, sensitive, and reliable, can provide a practical alternative to screen andquarantine a large number of animal samples within a very short period of time.4. To establish a rapid, sensitive ELISA to inspect antibodies to EHV-1/4, the equineherpesviruses 1 (EHV-1) gD gene encoding major neutralizing antigenic domain withcross-reactitve, conservative epitopes of both EHV-1 and EHV-4 was amplified by PCRassay and cloned into prokaryotic expression vector pGEX-6p-1. After induction oftransformed E. coli with the recombinant plasmid designated as pGEX-EHV-gD by IPTG,EHV gD was highly expressed in the form of GST-EHVgD fusion protein, as predicted43kDa in SDS-PAGE, which was about 32.7% of total proteins in bacteria. The sharedantigenicity of the recombinant protein was confirmed by Western-blot with antiseraagainst EHV-1/4. Hyperimmune sera generated in rabbits immunized with the expressedproduct could neutralize EHV-1/4 with high titers, indicating its good type-commonimmunogenicity. Following a serial treatment of denaturation, refolding, purification andPreScission Protease cleavage and re-purification of fusion protein with GSTrap FF affinity chromatography column, highly purified GST-gD fusion protein and GST-free gD protein(about 99%) were recognized specifically by both EHV-1/4 antisera in Western-blot. Theoptical dilution of antigen and test sera were determined by checkerboard titration before anindirect type-common EHVgD-ELISA was established and improved using theserecombinant gD fusion proteins (GST-gD) or GST-free gD (gD) as coating antigens,without significant divergence if at the same mol concentration. Horse antisera to EHV-1/4were revealed as positive reaction whereas horse antisera against equine arteritis, equineinfluenza and equine infectious anaemia were exhibited as negative reaction.EHVgD-ELISA was more sensitive with a detection rate of 41.1% compared to virusnueralization with a detection rate of 34.1%, but their conformance ratio was 93.1% and95.2%, respectively for tentative serum samples and clinical serum samples collected fromhorses for entry-exit inspection and quarantine. The agreement rate between home-madeEHVgD-ELISA and imported ELISA kit reched to 95.1%. To investigate the disseminationof Equine rhinopneumonitis in major horse populations from 5 provinces of North China,including Xinjiang, Neimenggu, Helongjiang, Jilin and Hebei, EHVgD-ELISA was used todetect antibodies to EHV-1 and EHV-4 in 22 198 horse serum samples, of which 3 821samples (17.2%) were identified as positive. Statistic analysis has shown thatseroprevalence was declining in June to the lowest level in October, and then climbing inDecember to the highest level in next April. This obervation may be explained byepizootiological characteristics of frequent infection or reactivation of EHV-1 and EHV-4 incold seasons of late winter and early spring. Morever, during epidemioiogical survey,seroprevalence in pasture raised horses in Xinjiang and Neimenggu was lower than that inintensively managed horses in Helongjiang, Jilin and Hebei, illustrating colse contactfacilitates virus transmission in horse population. Take together, the overall results suggestthat EHVgD-ELISA, which is rapid, specific, sensitive, convinient and reliable, can providea practical tool for serological diagnosis, epidemiological survey and entry-exit inspectionand quarantine of both EHV-1 and EHV-4.5. To develop a inspection system for EHV-1/4, standardized procedures for micro-serumcomplement fixation test (CFT), micro-serum neutralization test (VN) and EHVgD-ELISAhad been established to detect antibodies against equine rhinopneumonitis (ER), usingEuropean vaccine strain Rac H of Equine herpsevirus-1 (EHV-1 Rac H) as VN workingvirus, crude prepation of EHV-1 Rac H cultured in RK-13 as CF antigen andprokaryotically recombinant antigens within EHV-1 gD expressed in Escherichia coli as ELISA coating antigen. No cross-reactivity was observed in three above-mentionedserological methods with serum against other equine viruses, confirming their strongspecificity. Compared with the reference method (VN), CFT and EHVGD-ELISAdemonstrated 85.6% and 94.8% agreement, respectively, and CFT gave a relativesensitivity of 63.3% and a relative specificity of 91.5%, and EHVGD-ELISA gave arelative sensitivity of 100% and a relative specificity 93.4%. Linear regression of these VNand CFT titers as well as VN and EHVGD-ELISA titers revealed a highly significantcorrelation (R²=0.7008, p＜＜0.0005, student's t-test) and a more highly significantcorrelation (R²=0.7415, p＜＜0.0005, student's t-test), respectively. Of 8750 sera collected inhorses for epidemiological survey, in equid species for entry-exit quarantine frominspection and quarantine organs and some institutions outside the Chinese mainland(Kazakhstan, Hong Kong, Macao), seropositive reactivity were demonstrated in 1749samples in CFT, 1829 in VN, 2285 in ELISA. Using EHVGD-ELISA, seroprevalence wassubstantiated about 17.2% (3 821/22 198) in domestic horses from Xinjiang andNeimenggu in China and exported equid species.